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.

Dear Dr. Haneen nur,

Thanks for the valuable comment.

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...