Hi! I have a stable TCR transfected cell line but I realized I should also express CD8 alpha and beta chains in order to properly test antigen response.
One option is to go back to plasmid production and ligate my TCR chains into plasmids already containing CD8alpha or CD8beta chains, and then transfect cells with them.
But since I already have the TCR expressing cell lines, I wonder if I can just transfect CD8 alpha and beta chains into them.
Has anyone had any similar experience?
Hi,
I dont have experience on the cell line you mentioned, but I have done double transformation on Rosetta De3 cells. What I did was after the 1st successful transformation, I made the transformed cell into competent cells and retransformed them with another appropriate plasmid with different antibiotic resistance. Initial cotransformation didnt work, hence they had to be made into competent cells again.
I would probably test the easier method first, that is to transfect them with the CD8 alpha/beta, and if that doesnt work then go back to the plasmid production. Mainly it might effect the alpha/beta expression if they are not ligated in the same plasmid. That would be my biggest concern but if it work it works.
Hope this helps.
Regards,
Mikko Hynönen
Hi, Mara Llamas,
I have attempted to express human TCR a/b on cell surface of CHO cells, a non-T cell line.
I constructed a plasmid vector in which the human TCR a/b chains were combined into a single cDNA using the 2A self-cleaving sequence expressed by a CAG promoter.
On the other hand, TCR a/b forms a complex with CD3 and is expressed on the cellular membrane. Therefore, I purchased a mouse CD3 complex expression vector (Murine CD3 WTdelta-F2A-gamma-T2A-epsilon-P2A-zeta pMIA II) (Plasmid #52093) from Addgene, connected the mCD3 complex cDNA to another CAG promoter on another plasmid vector.
These TCR expression vector and mCD3 expression vector were simultaneously transfected into CHO cells.
As expected, human TCR a/b and mCD3 complex were expressed on the cell membrane, and their expression on the cell surface was detected by FCM analysis using an antibody for mCD3 complex (BD, Catalog No. 565643 APC Rat Anti-Mouse CD3 Molecular Complex).
However, we did not perform functional analysis of antigen recognition.
If you are using non-T cells, you will need to express the CD3complex at the same time.
I am not an expert about immunology.
My question is, why is CD8 necessary for the TCR-CD3 complex to function?
Does the TCR also require CD8 for membrane expression, or does the TCR only require CD8 for complexing with HLA?
Best,
Narumi
Hello,
I am not an expert in immunology but overall cotransfection is a common procedure. If you research cotransfection or double transfection, many protocols appear, so even though you are not doing them simultaneously, there doesn't seem to be a contra-indication into the quality of the transfections if you do them sequentially in the same cell. I would just maybe pay attention to how you design the "second" plasmids to make sure you can properly select the stable cell lines with both your plasmids. Hope it helps.
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