Dear Researchers Working in field of Tumor immunotherapy,
I am working with MC-38 colon cancer models. I had faced a critical issue when I tried to use TAA immunotherapy.
I have observed this phenomenon twice:
To study the TH1 specific response we are considering IFNγ and TNFα, When I am culturing splenocytes of tumor-bearing mice overnight with Antigen, I found there is a loss of CD8 cells. Is it very common phenomenon or I am making some mistake?
Awaiting for your crucial comments
Thanking you
Hi,
Please check your condition. Medium formulation and growth factors that you should use for CD8+ T cells propagation.
Regards
Tumor eversion mechanisms may be leading to the CD8 cell apotosis
Have you considered the fact that TCR stimulation leads to CD8 down-regulation on T cells? Have a look at CD5 as well. It usually goes up when such stimulation happens. I would also suggest you track T cells using cell trace or CFSE just to assess whether they are proliferation or dying.
Please define your co-culture conditions. Are you labeling antigen on total splenocytes? What are your APCs? Are you purifying CD8 T cells? If all cells are present together while you are labeling for the antigen, there is could be auto-lysis of CD 8 T cells; i.e. a CD8 T cell could be lysing other CD8 T cells in the co-culture.
Dear Dr. Banson Barugahare and Mehdi Mahdavi,
Thanks for the comment and suggestions.
Dear Dr. Moutih Rafei,
Thanks for the suggestion, I have not checked CD5, I will consider for next experiments.
Dear Dr. Anil Shankar,
Thanks for the kind comment. I am using total splenocytes not purified CD8.
The culture conditions are as follows:
Splenocytes of tumor-bearing mice were cultured for 18–20 h in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated FBS, 50 nM β-mercaptoenthanol, 100 units/ml penicillin/streptomycin, 2 mM l-glutamine (Welgene), and 20 μg/ml of antigen (5 × 106 cells/well). After incubation, GolgiPlug (BD Biosciences) was added for 6 h.
I agree with your point of autolysis here because splenocytes are from tumor-bearing mice which have got three immunizations.
I have never faced such problem when I am checking Th1 response in healthy mice with two or three vaccine doses.
So, my suggestion is label antigen on the APCs (total splenocytes in your case; I assume they are RBC-depleted) by incubating them separately from your effector CD8 T cells. Add the CD8 T cells to the culture once the antigen labeling on APC is done (about 2 hour incubation). What is your antigen, what peptide? And why do you call it s TH1 response? What you are doing is a CD8 T cell effector response.
Dear Dr. Anil Shanker,
I am using CEA peptide because my MC-38 cells are overexpressing CEA. Splenocytes are RBCs depleted using RBC lysis buffer.
I am studying IFN-γ and TNFα in CD8 and CD4 cells, that is why I called this Th1 response. Summary of the experiment is as follows:
These splenocytes after Golgi plug treatment were washed three times with ice-cold FACS buffer. Cells were blocked on ice for 30 min with an ultra - block solution. Cells then stained with FITC-conjugated CD4 or PEcy7-conjugated CD8 antibodies (BD Pharmingen). After surface CD4 or CD8 staining, cells were washed three times with ice-cold FACS buffer and subjected to intracellular cytokine staining using the Cytofix/Cytoperm kit. Intracellular IFN-γ and TNFα was stained.
I will try to check the method you have suggested to confirm the CD8 lysis, but how can I confirm the lysis. Do I need to stain these APCs before adding Effector CD8?
Thanking you very much for the suggestions.
Hello,
I worked on invariant natural killer T cells, and I found that these cells become anergic upon activation with a specific antigen and they down regulate their specific receptor. you may have the same situation with CD8 cells (anergy + down regulating their receptors). And this is not the case when isolating healthy NKT cells.
We have one related paper, hope this can help to find out few answers:
Article Application of radially grown ZnO nanowires on poly- l -lact...
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