I am planning co-culture experiments between activated T-cells and TNBC cancer cell lines. What sort of controls to use since a reviwer raised this issue of controlling for direct effects of MHC1 presented antigens on T-cell function in co-culture experiments.
Dear Jabed,
I just wanted to briefly send you some ideas for your experimental problem and your reviewer issues. I do think that there are two (and a half) routes you might wanna consider.
The first one one would be blocking the MHCI-TCR interaction with antibodies against the MHCI (HLA) molecule, which of course means you will have to characterize your cell lines for HLA allele expression and you may need different antibodies (and the respective controls) as a one size fits all solution (depending on your TNBC cell lines) will not be possible. A more elegant way would be to use a blocking antibody against the α3 domain of the MHCI molecule, which will prevent the CD8-α3 interaction and hitherto the MHC-TCR interaction as well, as CD8 stabilizes the MHCI molecule in the immunological synapse of cytotoxic T cells. You could equally well try an antibody against CD8 that blocks that specific interaction.
Another possibility that you might want wanna exploit is to turn off peptide loading on MHCI in your TNBC cells, by transfecting your cell lines with a viral construct that prevents peptide loading. Off the cuff I'd suggest ICP47 from HSV, which competes with peptides in the ER for TAP binding and loading onto the MHCI molecule. Please see the attached review for details.
Article The MHC class I antigen presentation pathway: Strategies for...
I hope that helps a little!
All the best & good luck with your experiment,
Michael
Thanks Michael for your suggestions. I have little experience in tumor immunology so To keep it simple can I use anti-cd8 antibodies to block this pathway? Also what sort of t-cells should be used in coculture with tnbc cancer cell lines? Is there any Atcc t-cell line to use in these cocultures? Thanks again.
Dear Jabed,
to be honest I actually doubt that there will be a lot of response in co-culture experiments between T cell lines from ATCC (which in itself are mostly derived from tumors) and TNBC cell lines (as long as they are MHC matched). If they're allogenic one may see something (especially if you make your T cells more aggressive), but that's just because of the HLA mismatch.
I would use T cells isolated from PMBCs (that are HLA matched with your TNBC cell line) or even better T cells from your breast cancer patient that are isolated from the tumour directly. Only in that case it makes real sense to assess the reactivity of your T cells towards tumour derived antigens presented via MHCI as these clones should have encountered these in secondary lymphoid organs. I would then use the following antibody (which blocks presentation via MHCI according to manufacturer), however it only works for HLA-A,B,C alleles.
Here's the antibody:
https://www.biolegend.com/fr-fr/products/purified-anti-human-hla-a-b-c-antibody-1874
I am not sure about the exact clone to use for block of antigen presentation via MHCI with anti-CD8 blockade though the attached paper outlines the critical amino acids, so with a little searching for anti CD8 epitopes encompassing these amino acids you might figure a suitable antibody.
http://www.jbc.org/content/280/30/27491.full.pdf
I hope that helps for a start. Unfortunately, there's little more I can do as I don't know the specifics of your experiments.
Good luck,
Michael
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