I want to assay if addition of a cytokine is able to prevent exhaustion of CD8+ T cell. I cultured CD8+ T cells with anti-CD3 anti-CD28 during 24hs with/without the cytokine and after that I measured PD1 expression but I had problems with the induction of exhausted CD8+ T cells. Are there another way to do this experiment?
One way to induce CD8 exhaustion is LCMV infection.
Are you using plate bound anti CD3 and soluble CD28?
Culture of CD8 T cells on irradiated feeder cells (irradiated PBMCs) with high levels of IL2 (6000 units/ml) makes them proliferate massively and you could detect exhaustion markers on these cells. PD1 (early exhaustion), Lag3 (middle) and Tim3 (late stage exhaustion) markers can be detected. (Check our rapid expansion protocol (REP) odor expansion of TILS from Rosenberg lab and just apply that in your experiment. Best!
Thanks! We dont have LCMV infection in our lab. We used plate bound anti CD3 and anti CD28 (non soluble). Thanks Anchana and Andre! Next week we will assay different condition and tell you the result! Thank you very much!
hi,
Reading your set up, I was wondering if 1 day post activation you noted any different expression pattern for other receptors than PD1, such as Tim3 or CTLA-4?
Moreover, was 24 hours stimulation enough to induce a visible response on PD-1 expression?
Thanks in advance :)
Just in case someone else will be interested: add Il-2, reseed the cells onto freshly coated plates 3-4 times each 2-4 days.
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