I give high does(500ug, i.v.) ova peptide to induce OT II mice in vivo, but this method doesn't work. And I still don't know why. I would be very grateful it anyone could tell me how to do this experiment.
Dear Yang,
Could you elaborate on your protocol (how many T cells you transfer, and at what time/which organs you look at ?
On day 0 and day 3, I give OT II intact mice 500ug OVA peptide, and give control group PBS. On day 10, sacrifice the mice and adoptive transfer the CFSE-labeled spleen OT II cells from OVA-treated mice and PBS-treated OT II intact mice to normal B6 mice. One day after transfer, I restimulate the transfer OT II T cells by i.p. immune the recipient B6 mice with 50ug OVA peptide plus CFA. 3 day after immunity, take out the spleen and see transfer OT II T cells proliferation( use CFSE), but these two groups proliferate equally, which means I didn't induce anergy successfully.
You could look at some H. Pircher papers from the 90's on this peptide tolerization strategy.
Do you administer the OVA peptide i.p. or s.c.? Are you emulsifying the OVA in IFA during tolerization? You could always add another treatment, but I don't think that would be necessary to see a difference in proliferative capacity.
@Cory J Knudson ，I administrate OVA peptide i.v. and I just use OVA peptide to immune mice. A lot of papers say that proliferation and cytokine production are the two indexes which used to detect anergy cells. So, could you please tell me why you think it's not necessary to see a difference in proliferative capacity.
Sorry if that was confusing. I was just stating that a third OVA peptide administration should not be necessary to see differences in proliferative capacity.
As I stated above I would try giving the OVA peptide i.p. emulsified in IFA (which has been published before). It seems that forming these depots of antigen helps induce tolerance.
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