Recently, I want to verify the certain treatment enhancing on antigen presenting ability of macropohages.
I searched the literitures, using OT.II T lymphocytes and OVA antigen, OT.1 lymphocyte and OVA antigen are often chosen to verify the ability on CD4 and CD8 T lymphocyte differentiation and proliferation.
I want to konw whether there is any other method to do the experiment. Because we do not have OT.II or OT.I mice.
How about co-culture the Naive T cells and macrophages in the presence of certain antigen "X". If this method is acceptable, what antigen "X" could be？
ps: Infectious and malignant diseases like tuberculosis and lung cancer are our intersted field.
It is difficult to answer your question without knowing what your readout of CD4 and CD8 differentiation and activation is. The reason that OT.I and OT.I mice are used is that these mice carry transgenes that specify the T-cell alpha and beta chains specific for OVA displayed on I-Ab, the class II MHC molecule of C57.Bl/6 mice. That means all of the T cells from these mice - both CD4 and CD8 - will react to the antigen, so you can get the kinds of answers you are looking for.
Getting naive CD4 and CD8 T cells to react to a foreign antigen is no big trick. You need antigen-presenting cells - in your case, macrophages that have been loaded with your antigen of choice, be it OVA or KLH or whatever protein you like, of the same MHC type as your T cells, add the CD4 and CD8 T cells, incubate together for X amount of time, and measure proliferation.
This was done in the past by 3H-thymidine incorporation. Now, dyes such as MTT are more often used. The problem for your model is that since the cells that respond to your antigen are rare - 1:100,000 vs every cell in the transgenic system - it may be extremely hard to see the effects of treating the macrophages on T cell proliferation. That is likely why the transgenic system is used.
I am presuming you are assessing the effect on CD4 and CD8 T cell differentiation by flow cytometry. That would be straight-forward in the transgenic system, but much harder in the naive system, since the responding T cells are rare. It may be possible, I just don't know the state of the technology in that field.
A follow-up idea: one way to look at a larger portion of the T cell population and response would be to use a superantigen, such as staphylococcal enterotoxin B [SEB]. Superantigens bind class II MHC on antigen-presenting cells and to multiple Vbeta regions on the TCR. I cite SEB because it binds to I-Ab on C57Bl/6 mice. The problem here is that your readout may only be detectable in CD4 T cells and not CD8 T cells, but I do know that SEB on C57Bl/6 APC triggers a significant proliferation response by T cells.
Dear Fengshuang Yi, did you tried the method with SEB, that Gary Lee Gilmore suggested? Did it work? Thank you for the suitable question, recently I faced with the same problem.
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