If you want to check the frequency of virus-specific CD4 and CD8 cells, there are two ways:

First one is FACS:

You need to stimulate your PBMCs with specific protein of your virus then check the frequency of CD3+, CD4, and CD 8 cells.

Another method is ELISPOT:

You don’t need to stimulate your PBMCs just use magnetic beads specific to CD4 and CD8 coat your plate with a particular virus protein and follow the ELISPOT protocol. You can use different kind of cytokine antibodies specific to these cells.

There is another method, but this can only give you an assumption, if you have proper controls, I mean healthy volunteers. You can check the total cell frequency in healthy and infected volunteers, and then you can calculate the approximate change in T cell population.

The process you have mentioned is not for quantification that is usually used for cell proliferation.

I hope this can help.

All the very best

Prashant Sharma,

Thanks for your detailed suggestions. I've never done ELISPOT before so it didn't come to my mind. I'll see what and how I can do with it.

As for the FACS method, I'm still a little confused. By stimulating the cells, surface marker expression and cytokine production will be analyzed by a cytometer and tell us that to what extent the antigen-specific T-cell responses are generated, right? I thought cell proliferation after stimulated with a specific antigen/pathogen could (to some extent) give us similar results or conclusions. Do I misinterpret this?

1. This is doable but not easy. Better methods are available (see below).

2. You will have very few DCs if you try to isolate them. Monocyte derived DCs would be more practical but less physiological.

3. 2 mL of blood would give you roughly 2 million PBMCs, out of which very few would be DCs.

I would not recommend testing on your blood. Try and see if you could source PBMCs from someone who has had an acute/ chronic infection with this virus and do your optimization experiments with these cells. Many vendors these days provide primary cells.

Better methods:

If you want to analyze the frequencies of virus-specific T cells you can do two things, as Prashant suggested.

1. Intracellular Flow Cytometry: Put PBMCs in culture with viral antigens and then stain the cells with CD3/CD4/CD8. You should also include functional markers like IFN-g, TNF and CD107a to identify virus-specific T cells that respond to the antigens.

2. ELISPOT: More sensitive than Flow Cytometry. Buy an ELISpot kit (Mabtech ones are really good) and follow the manufacturer’s protocol. Basically, coat the plate with the relevant coating antibody, plate your PBMCs in the presence of viral antigens. Develop the plates and any spots you see would be virus-specific T cells that responded to the antigen.

These two techniques would give you the frequencies of functionally responsive antigen-specific T cells.

If you want to look at proliferation of T cells in response to viral antigens, you could do the following.

1. CFSE staining: Culture CFSE stained PBMCs in the presence of viral antigens. At D6-8 post culture, do flow cytometry to look at dye dilution as a measure of T cell proliferation.

2. Ki-67 staining: Put PBMCs in culture with viral antigens. At D6-8, stain the cells with CD3/CD4/CD8/Ki-67 to identify antigen-specific T cells that express Ki-67 (proliferating cells).

In my experience, Ki-67 staining works better than CFSE when looking at very low frequencies of proliferating cells.

Hope this helps.

Dear Chao-Wei Yeh,

Your interpretation is correct nothing is wrong in it, you can also go for cell proliferation and cell frequency check, but it would be difficult to interpret and analyze the results.

My recommendation would be to follow as Julie Joseph has suggested, it would be easy to interpret the data you just need to go with two experiments separately.

All the very best

Julie and Prashant,

Thank you very much. This really helps and I learned a lot!