I am trying to induce passive EAE by adoptive transfer of primed T cells from WT C57BL/6 donors to WT C57BL/6 recipients. The protocol I followed was:
Day 0 - immunised donor mice with 200ug MOG35-55 s.c. + one does of 200ng pertussis toxin i.p.
D 8 - sacrifice donors and remove lymph nodes and put cells in culture with 20ug/ml MOG + 10ng/ml IL-23
Day 11 - blasting cells observed. Transferred 10 million cells i.p. per sub-lethally irradiated WT recipient (irradiated approximately 3 hours prior to cell injection). =D0 of scoring EAE.
Now it is Day 15 post cell transfer and my mice are not displaying any EAE symptoms. I see some protocols also give pertussis with the cell injection into recipients and again two days later? Or did I inject too few cells? What could the problem be?
I hope our publication will be providing informations you requested.
Unfortunately not since I am using C57BL/6 mice and you used SJL mice in this publication. But thanks anyway!
This protocol works very well. It requires PT injection on the day of transfer and 2 days after. Cells are administered by intra peritoneal injection. Disease can be induced even with sub cell optimal numbers.
That's because Th2 polarized cells (IL23 stimulated) do not induce EAE w/ MOG peptide! And if they do in the sub lethal irr mice it will be a milder form, with limited clinical manifestation. The only reference I've seen for Th2 pol. cells inducing EAE is with MBP, not MOG, and the clinical symptoms take longer to develop.
Below is a procedure we routinely use, with robust disease.
Procedure:
day 0:
Mice are anesthetized and injected subcutaneously with a total of 200μL over two areas
(between shoulder blades, above right hip) with 3 mg/ml MOG35-55
mixed 1:1 with 3 mg/ml Mycobacterium tuberculosis in Incomplete Freund's Adjuvant on day 0.
(300 μg peptide and 300 μg Mycobacterium tuberculosis [H37Ra])
Two hours later, mice are injected intraperitoneally with 100 μL of 300 ng Pertussis Toxin.
day 2:
Mice will be injected intraperitoneally with 100 μL of 300 ng Pertussis Toxin.
day 9:
Draining LN (DLN) cells are collected
Polarization of lymph node cells
day 0: (7 days after culture)
Prep of polarized cells.
Cells adoptly transferred i.v. into naive C57Bl/6 mice @ 15x106 cells/ms
So Elizabeth, how do you polarize your cells before transfer? And how do you keep them in culture for 7 days? That is a long time...
We stimulate with MOG and IL23 to induce Th17 recall response, not Th2.
IL23, MOG anti IFNg, and anti-IL4. I don't do the ex vivo stim. If you need more specifics, let me know, and I'll get my collegue's input.
Administer PT to your recipients (200 ng/mouse/day) on days 0 and 2 post-transfer ip but not your donors as you want to keep your donor cells in the LN and spleen.
Consider checking your donor cell cytokine profile on day or transfer to ensure your IL-23 is working (good to include a small culture without IL-23 as control).
You could also leave the donors another day or 2 (only if you haven't given them PT) - we would normally harvest between 9-12 days post immunization Our protocol is listed here but feel free to email me if you need more details:
http://www.jimmunol.org/content/190/7/3225.long
Thanks Denise. But if you administer PT to the recipients upon transfer, do you also sub-lethally irradiate them before transfer?
@Shane Russell: In your protocol you culture cells in TH1 and TH17 conditions. Do you transfer both cell types 50:50 into recipient mice or do you transfer only one cell type? And do you think the protocol would also work in a smaller scale using e.g. 96- or 24- well plates?
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