I'm trying to differentiate purified (>95%) human naive CD4+ T cells in vitro. This topic has been addressed several times in various settings, but I wanted to get thoughts on specific “tricks” gain by experience. I’m using these conditions:
1. 1x10^5 naïve human CD4+ T cells isolated using StemCells’s naieve CD4 kit
2. 1ng/mL IL-2, 20 ng/mL IL-12, 5 ug/mL anti-IL-4. Both cytokines from Hemacare, blocking antibody from R&D.
3. Culture in 96-well round bottom for 3 days
4. 1:2 ratio of beads to cells. T cell activation beads from Miltenyi, CD3/CD28.
Analysis on d7 by intracellular staining showed very little T-bet, IFNg cells. There were large amounts of IL-4. Any thoughts? I’m currently titrating and testing the antibodies and intracellular protocol. Reactivation on d7 is by PMA/iono for 6hrs with Brefeldin.
We have found in the past with T-cell polarization that 7 days total seems too short for a robust polarization and that the Blocking antibody needs to be replenished often. T-cells are very stuborn and require quite a bit of coaxing to stay going "in the right" direction.The Dynabeads we moved towards after this studymuch like you are doing they work fantasticand you can easily replenish the media and not lose them with the magnet. Hope this helps
Our following protocol worked well.
Epigenetic repolarization of T lymphocytes from chronic lymphocytic leukemia patients using 5-aza-2′-deoxycytidine
Jason A. Dubovskya, b,
John J. Powersa, b,
Yang Gaoa, b,
Luis F. Mariussoa, b,
Eduardo M. Sotomayora, b,
Javier A. Pinilla-Ibarza, b
2.2. Cell culture, drug treatments, and T cell polarization
Unless otherwise stated cells were cultured in vitro at 37 °C and 5%CO2 using RPMI1640 medium supplemented with 10% fetal calf serum and antibiotics. Drug treatments were carried out on plate-bound anti-CD3, soluble anti-CD28, and IL-2 (20 U/ml) stimulated magnetically isolated T cells (>95% purity) for 72 h in complete medium using the indicated concentration of 5-aza-2′-deoxycytidine (5A2) (dissolved at 10 mM in DMSO). At the conclusion of treatment cells were washed twice using pre-warmed serum-free RPMI1640 and subjected to the various assays (Supplementary Fig. 1). All CD4 T cells were negatively isolated using magnetic separation (EasySep CD4+ and Naïve CD4+ T-cell enrichment kits, STEMCELL Technologies, Vancouver, BC, Canada) according to the manufacturer protocol. Th1 and Th2 polarized T cells were obtained from magnetically purified naïve human CD4 T cells by weekly stimulation with plate-bound anti-CD3, soluble anti-CD28, and IL-2 (20 U/ml) in the presence of IL-12 (10 ng/ml) and anti-IL-4 (1:100) for Th1 or IL-4 (5 ng/ml) and anti-IL-12 (1:100). Medium was replaced twice weekly and stimulation was repeated for four weeks upon which a sample was collected and tested via CBA array and qRT-PCR for polarity (Supplementary Fig. 2).
Thanks John. I've seen the statement "stimulation was repeated", but I'm a little unclear. The cells are maintained with anti-CD3/CD28 in the plate, so what does repeated stimulation refer too? I think I'm just missing an obvious point.
If you work with pure naive T cells, there is no need in adding anti-IL-4.
I have cultured them before to induce Th2 differentiation, and always took Th1 differentiation along as positive control.
I simply cultured isolated (pure) naive CD4+ T cells on anti-CD3-coated plate in presence of anti-CD28 (as described in attached publication). And added 50 ng/mL recombinant human IL-12. Incubate for 5 days, then restimulate to look at cytokine production ICS. More than 50% of the cells will produce IFNgamma.
Article Activation of human basophils by combined Toll-like receptor...
I just saw your question
1) T cells recieved weekly stimulation with plate-bound anti-CD3, soluble anti-CD28, and IL-2 (20 U/ml) in the presence of IL-12 (10 ng/ml) and anti-IL-4 (1:100) for Th1 or IL-4 (5 ng/ml) and anti-IL-12 (1:100).
A) Medium was replaced twice weekly with the cocktail below (IL-2 (20 U/ml) in the presence of IL-12 (10 ng/ml) and anti-IL-4 (1:100) for Th1 or IL-4 (5 ng/ml) and anti-IL-12 (1:100)) (we removed media without removing cells from the well)
B) Stimulation (using plate-bound anti-CD3, soluble anti-CD28 later we used dynabeads) was repeated for four weeks (Each week we removed cells from one plate and added it to fresh one with (plate-bound anti-CD3, soluble anti-CD28 later we used dynabeads)
So essentially cytokine cocktail 2 times a week and CD3-Platebound with CD28 once a week
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