As part of an effort to determine whether a certain proliferative response of virtual/innate memory T cells (CD44hi) I tried to knock-out the TCRa of these primary T cells and to culture them with my stimulation of question or with what I thought to be a TCR-independent stimulation, PMA/Ionomycin. I wanted to then, after 96 hours of culture, check for the ratios of TCRb-positive vs. TCRb-negative T cells in both cultures, assuming the PMA/Ionomycin stimulation will, on one hand, keep those primary T cells alive and well during those 96 hours, but on the other hand will get them to proliferate in a completely TCR-independent fashion. This does not seem to work, however, and more than 90% of the cells in that culture die (should I try it without ionomycin maybe?).
My questions are, therefore:
1) What would be a proliferation-inducing stimulus which I can use for T cells and which is practically/completely TCR-independent? As these are innate/virtual memory CD8+ T cells, maybe I should use IL-15/IL-7/both?
2) Do you have any suggestion as to how I shall examine the TCR-specificity of a reaction of innate/virtual memory CD8+ T cells without manipulating MHC presentation (KO of b2m from the immunogenic cells gave ambivalent results, which is the reason I wanted to check the other side of that coin, the TCR-dependency)?
I would really appreciate any effort to help!
Dear Omer,
I love your question because it already contains parts of your thinking process, which is really great.
I would be curious to know why you consider PMA/ionomycin as not being a TCR-independent stimulation, as I thought it was more or less activating the same signalling cascade, just bypassing the specific TCR signal. Here is a discussion in RG about this.
I would have suggested to induce a "bystander activation" but I realized you considered already IL-15 and IL-7. Maybe you could also try co-stimulations with anti-CD40 and IL-2, something like this ?
https://www.researchgate.net/post/Unspecific_stimulation_with_PMA_ionomycin_vs_PHA_for_intracellular_cytokine_staining_of_T_cells
If you wanted to use PMA/Ionomycin you should be ok to stimulate for 96hours without too much impact on cell viability, however if you leave them for any longer than this then the cells will most likely apoptose.I understand what Julien says about PMA/Ionomycin stimulating the same pathways as TCR stimulus, however costimulatory receptor stimulation can stimulate these pathways too. We use it as a positive control in our assays, and also to determine the intrinsic cytokine production capacity of T cells whilst discounting the contribution of TCR signal defects.
Watch out for TCR down regulation though as strong stimuli like PMA/Ionomycin can mediate down regulation.
Dear Omer,
I think you might be running into 2 potential problems, although I cannot say for sure because you did not specify the acutal concentrations you are using in your assays.
1- Too strong stimulus: Maybe tamper down the PMA/Iono stim. It is generally very potent and it bypasses many cell surface events/molecules. You might be inducing cell death more than proliferation. e.g. for the in-vitro generation of certain "lineages" it is actually better to use low anti-CD3, like in the case of iTregs.
2- Antigen experienced/mature T cells (like your CD44+) are notably less prone to be stimulated through the TCr in this kind of in vitro assays compared to naive T cells. I would strongly reccomend for you to add a positive control (naive cells from same donors) and also some other controls (e.g. tirtiate the PMA/Iono best conentration and IL/15/IL-7 combo as well). I think the IL-7/IL-15 idea might work well. Check what is used in ILC or other innate cells cultures that could be a best combination.
Viel Glueck!
Oh, by the way, I would also consider adding IL-2 to promote survival/proliferation in a TCR-independent manner.
Dear friends,
Thanks for the fantastic replies!
Julien: I think I was mistaken; I had some weird results arising from the usage of PMA/Ionomycin and then read somewhere that they may still require, indirectly, involvement of the TCR/CD3 complex. However, I didn't read it through and now I think it was probably not the case, as I realized my results seem to arise from massive death my cells undergo due to the treatment with PMA (and probably regardless of Ionomycin). BTW, I do constantly use IL-2 in the medium (it doesn't seem to cause significant proliferation of untreated memory cells by itself).
Emily: I do use it just for 96h. However, I think I used to high of concentrations, as even after 48h I witnessed significant death. This death is especially problematic as it seems to come on top of death induced by loss of the TCR.
Damien: I think it's indeed the strength of the stimulus, and thus I will next try to calibrate it. Also, I do use IL-2 in the medium in any case :)
Werner: I did think of PHA or Con-A, but... I thoguht they work by cross-linking TCRs, so... Wouldn't they enrich for the non-KO cells?
Hi Omer,
You will get a lot of cell death with PMA/Ionomycin. When I most recently did proliferation assays I had to stop this at 4d and the other stimulus (PHA/TAE) at 5d for this reason. PMA activates PKCtheta and ca2+ mobilisation so is downstream of TCR/CD3, whereas PHA is a lectin which cross links all cell surface proteins including TCR/CD3 and other cytokine/costimulatory receptors.
You won't require IL2 in the medium unless you are doing TCR/CD3 +/- IL2.
I now utilise a death marker called zombie dye to gate out the dead cells, as dead cells can lose staining with CFSE or CTV, and look like a divided population.
I hope this helps.
Emily S J Edwards Do you have a specific protocol for this stimulation with PMA/ionomycin with specific concentrations? I am wondering whether to replace these reagents daily/change to fresh non PMA/iono media. Thanks for your help!