If I am understanding you correctly you are planning on having antigen's from the virus presented to the CD8 T cell via a class 1 MHC. Are the MHCs going to be mismatched if yes it will be difficult to confirm cells are antigen specific.

Why don't you use the CD8 negative fraction from your T cell isolation(depleted of CD3 T cells of course) as a source of autologous APCs.

Regarding Radiation doses typically cancer cells lines get high dose radiation like the 5000 Gy you mention. Primary autologous cells typically get a lower dose around 20-100 Gy. Run a well without your T cells and see if the accessory cells proliferate.

Hi William Suslovic , your understanding is correct. MHC matching was the main reason we chose the P815 immortalized masts (to match the BALBc mouse haplotype "d", which will supply the CD8s). Is there anything else that should be cross-matched? I'll also have four controls: (1) co-culture of mock-infected masts with CD8s, (2) co-culture of mock-infected masts with CD8s stimulated by CD3/CD28, (3) infected masts only, and (4) CD8s only stimulated by CD3/CD28. If the first control (mock infected co-culture) has *a lot* of death, then that may indicate mismatched haplotypes--although I'm not sure what to do if that happens. Is that enough controls, am I missing something? Or is that too many? I feel like you can never have too many controls...

The main reason against using the CD8 negative splenocyte fraction as APC is time constraints. I've never worked with primary immune cells before and I'm afraid they're going to die really fast.

From my understanding, once the CD8s are purified from the spleen, they should be incubated with proliferation signals immediately, correct? So if we want an antigen-specific proliferation then that antigen has to be present on day 1. But it would take at least a day for the mast cells (or CD8- APC fraction) to process the viral infection and present antigens on MHCI. So I'm worried that time lag may be just long enough to kill the CD8s. But this is also my first immunology experiment, so I really don't know how valid my concerns really are.

Thanks so much for all your insights!

What is your goal? Just expansion of antigen specific CD8+ lines?

Even with the cells HLA matched the Cell line may present neoantigens to the CD8 T cells since the intracellular proteins of the immortalized cell might be different than of your mouse. That's not to say it won't work of course using immortalized cell lines is quite common, though typically autologous immortalized cells for antigen presenting cells.

The closer matched HLA the better inbred mice certainly have less HLA diversity than people so if the Mast Cell line came from the same strain it is more likely to work. As for other HLA antigens you can check one of the alloantigen lists: https://www.biolegend.com/Files/Images/media_assets/support_resource/BL_MouseAlloantigens_041116.pdf

The Controls you have seem fine I organized below for my understanding.

1. Mock Co-culture no TCR Stimulation: Controls for non-specific activation(HLA or other accessory cell effect)

2.Mock Co-Culture TCR stimulation: Postive control + Co-Culture control

3. Infected Mast: No T cell control(can be useful to confirm successful radiation and infection)

4. TCR Stimulation: Positive T Cell only control(shows cells can proliferate)

I think you can do without the Mock Co-Culture with TCR stimulation(2) if you had to drop one. I'm not sure what it is controlling for that your other controls aren't already covering. Depending on your methods and goals you could loose more of them ie you can loose control 3 if you can prove infection/successful radiation treatment in your co-cultures.

Could be interesting to see what happens with a immortalized Mast cell for co-culture. You would have to prove the T cell specificity to viral antigens of course. I think you would get cleaner data from autologous cells.

As for working with primary Immune cells they should be fine to leave overnight or 24 hours after isolation in complete media. Human T cells will sit there just fine for a little while, even longer if you give them Il-2 or some other survival factor that you will probably give them later anyway(might affect differentiation though!!). I'm not experienced with mouse T cells but I Imagine short term survivability isn't too different.

Furthermore depending on how long you have to infect your cells for you can incubate them with the virus for the necessary time for infection, wash them and then immediately go to co-culture while viral proteins start to be expressed.

I can envision a workflow where you homogenize Spleens, Isolate CD8(+) cells leave over night, Irradiate CD8(-), Infect overnight then mix the next morning. Alternatively use the previous mouse's CD8(-) Cells for the next mouse's CD8(+) expansion. Just freeze down the CD8(-) fraction when you get it(quickly as subpopulations will adhere to plastics and you will loose them) and then use those frozen cells infection and presentation to the next mouse's CD8(+) cells. That way you can prepare Infected cells the day before assuming freeze thaw doesn't affect infection rates.

No mater the method as long as you confirm the antigen specificity at the end of your experiment it may not matter how you got there.