I would like to compare kinetics of CD4+ T cell activation among various species (mouse, cow, pig, dog, cat). I wonder what common stimulation procedure to apply to these T cells (CD3, PHA, sAgs?) and what could be a best output parameter (synapse formation, CD69, CD62L, IL-2?) for comparison. Is there any published data showing the kinetics of CD4+ response in the cow in comparison with the mouse or man?
Hi,
I think that CD3/CD28 cross-linking/immobilization would be a good start but I cant help you with the specifics.
In terms of the markers, they do generally correlate with or are part of different steps of the activation cascades or effector functions. So they representing different steps and it really depends what you want to measure. For example CD69 you should see much before IL-2. The surface markers will be easier to measure than for example intracellular cytokines. Another option would be to do QPCR for cytokine genes.
Also you need to be aware of your starting populations, naïve, central and effector memory CD4s will respond differently, that should be controlled.
Good luck,
Hannes
If these are naive and uninfected cells, usually a culture with anti-CD3 and anti-CD28 is the best model for T cell activation. Early in the culture the cells will activate surface markers and secrete cytokines, so you could look at surface CD25, CD69, or HLA-DR/CD38 expression and IL-2 or IFNgamma. If you extend the culture the cells will proliferate, so you could also check for cell proliferation via CFSE dye dilution. If you have enough cells available you can follow these responses over multiple days to establish kinetics.
That is a tricky question, as good mitogens for one species may be useless in another. Activating antibodies to a certain species may not interact at all with T cells from other species. You need a stimulus that activates all of the T cells equivalently. An option is to use allogeneic stimulation: CFSE label your responder T cells and stimulate them with allogeneic PBMCs of fully mismatched animals. The more divergent the strains in the species, the most powerful the immune response. Concavalin A may work, but I have the feeling it is far better in human PBMCs than in mouse, and PHA doesn't work in mouse T cells at all.
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