3 replies to this topic

[Doc J]

Hey All,

I'm new to class II Ag presentation and can't seem to find any protocol source for pulsing mouse macrophage cells with exogenous peptide fragments to load the surface MHC-II with a specific epitope. I've done this many times for a class I assay in which I would incubate a different cell line with the exact fragment length for display on a class I MHC molecule, which would then simply displace whatever epitope was being displayed. This worked quite well. However, I have tried similar methods with a class II epitope but I'm unsure if the negative results I've seen are due to problems with my antibody (yes there is an antibody that binds to MHCII with peptide), or peptide loading, or both.

Any help would be appreciated.

-J

[Doc J]

I'm looking into two cell lines: macrophage-like and a dendritic cell derived. Literate seems to indicate macropinocytosis is one way of getting fluid-phase antigen into MHC-II pathway, but I haven't found any specifics on amounts of antigen to use or activation state of the cells.

While the information I have come across seems to indicate that some MHC-II is present on the surface even as an immature/resting cell, I'm still looking for literature that estimates the number of MHC-II molecules on the plasma membrane at this time. I had already done some experiments that have determined that MHC-II is detected on the cell surface of my cells, but this still does not answer the underlying question that started this topic. Thank you again for anyone willing to help.

[Doc J]

BUMP

Ok so this topic is not dead, but responses seem to be lacking. I'm still looking for help on this. I've confirmed many times that a log level of difference can be detected of MHCII+ vs MHCII-/unstained cells. Any ideas of something for a postive control for flow cytometry?

[Astarte Biologics]

Doc J, on Dec 16 2009, 09:16 AM, said:

BUMP

Ok so this topic is not dead, but responses seem to be lacking. I'm still looking for help on this. I've confirmed many times that a log level of difference can be detected of MHCII+ vs MHCII-/unstained cells. Any ideas of something for a postive control for flow cytometry?

I think that the problem is the difference between MHCII and MHCI.  I've had no problem getting peptide onto macrophages or DC or other cell types by simply incubating cells with peptide for 1 hour at 37degC.  My read out though is recognition by antigen specific T cells, not antibody.  I've looked at class I MHC stabilization on the cell surface using staining and it sounds like you have too.  I don't know if you used the same system I did.  I used the T2 cell line which lack the antigen transporter.  HLA-A2 gets to the surface with a  peptide but it is easily displaced.  It is also not stable and if you treat with brefeldin A you can watch it decay off the cell surface.  MHCII is less dependent on peptide for stabilization and a lot less picky about what peptides will bind.  Peptides hang out of the binding pocket and can be a wider range of lengths than MHCI peptides.
I'm not familiar with the antibody you are using but I would think it would be hard to detect the fraction of specific MHCII-peptide complexes amongst the majority of MHCII with other peptides bound.