Hi, everyone:
I want to determine whether IL-2 and IL-15 have different effects on immunized CD8+ T cell effector function.
In this study, I immunize WT mice with peptide on day 0 and 7, and harvest spleen on day 14 post immunization.
For investigation of cytokine effects, I purpose to re-stimulate splenocytes with peptide and subsequently treat with IL-2/IL-15 in vitro, and analysis of IFN-γ expression via intracellular staining.
Does anyone have any recommendation for in vitro stimulation protocol?
Detailed methods such as time duration of peptide re-stimulation and cytokine treatment? What time points should I harvest cells for analysis?
Look forward to your replies!
Hi!
I performed LCMV infection model. There, everything was easy and straightforward. Day 8 post infection, you collect splenocytes and add the peptide of interest (1 mcM is a good concentration to start with) together with BfA from Biolegend. 6 hours later, you just stain live/dead and surface markers, fix, permeabilize and stain with anti-IFNg.
I used 5-6 millions splenocytes per well in round-bottom 96-well plate, because I required a rare CD8 T-cell population and the cell losses during washing steps are just horrible. Probably, you will face the same problem.
I haven`t tried cytokine stimulation along with peptides, but in my case, I believe, it wouldn`t affect the outcome significantly. But you can try. For CD8 T-cell expansion 50 ng/ml (100 ng/ml is better) of IL15 is good to start. I used 50-100 U/ml of IL2 for expansion. This is a bit strong (most of the protocols use IL2 at 20-30-50 U/ml for culture with peptide), but that`s I think what you need in your case.
If you plan to culture splenocytes with peptides for several to expand your cells then the situation will be slightly different .
Hello, Kuan!
You look at this protocol, please:
https://www.jimmunol.org/content/169/9/4928
Spleen cells were harvested from OT-1 mice (Ly5.1), prepared as a single-cell suspension, and lysed with ACK buffer to remove RBC. Subsequently, ∼2.5 × 106 spleen cells resuspended in 300 μl HBSS were injected i.v. into unirradiated C57BL/6 (Ly5.2) mice. Mice were then rested for 2 days and then vaccinated with ∼1.4 × 106 Ag-pulsed or sham (unpulsed) DC resuspended in 400 μl. Following vaccination, mice received i.p. injections of cytokine (0.5 μg human IL-15 or 0.2 or 1 μg mIL-2) every 12 h for 7 days. Recombinant cytokine was diluted with HBSS into 100-μl doses prepared fresh for each injection. In some experiments mice were injected with vehicle alone as a control.
In line with above answers, you can find a detailed protocol from one of my papers https://www.nature.com/articles/s41467-018-04832-2
and can adapt to your settings. Some other papers regarding CD8 T cells and stimulation from our group might have even detailed protocol. We used LCMV-GP33 to stimulate and it is a very strong antigen hence we can observe nice production of IFN-g and TNF-a and some Interleukins. You might want to do a kinetics to get a best timepoint for the re-stimulation or may be its already mentioned in the literature for the peptide of your interest.
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