Hello and Thanks in advance.
I’m fairly new to Biology so please bear with me here.
I’m currently in the process of designing an experiment using ELISPOT assay to detect antigen specific CD8+ T cells isolated from vaccinated mouse spleen. The peptides I’m stimulating the cells with are predicted to be MHC class I restricted just like how the reference papers suggest. However, I do not understand how a simple treatment of purified peptide would end up being presented by the MHC class I molecules by the antigen presenting cells. My understanding is that exogenous proteins are usually presented by the MHC class II molecules, therefore are targeted by CD4+ T cells.
Are there free “empty” MHC molecules that are just sitting on the membrane of the APCs that readily loads the treated peptides? But I thought class I MHCs need to have peptides bound in the ER in order to mature/fold properly and transported to the cell membrane.
Do the peptides go through endocytosis and then get released into the cytosol to go through the regular process that other degraded peptides go through? (Transported into the ER via TAP then get loaded onto the MHC)
I hope my question makes sense to some degree.
Again really appreciate the help.
Would also very much appreciate any other comments regarding ELISPOT assays for future tips :) such as why are DCs used as APCs when most nucleated cells are use MHC class I molecule.
The short answer is yes, MHC I can exchange peptides without going through the entire peptide loading process. And also, endocytic vesicles are leaky, so there is a good chance the peptides will lead into the cytosol.
Although truthfully, I'm not sure anyone really understand exactly how loaded peptides work. Because we do the same with MHC II peptides too. Basically, I think the assumption is if you start with the restricted peptide, you have a better chance that even if it goes through the normal process, you will end up with presentation with the peptide you loaded.
Frist of all thank you very much for your input Dr. Hwang!
It is rather disappointing to hear that my question can't be clearly answered yet.
I've been reading up on "cross presentation". If your suggestion of leaky endocytic vesicles freeing up the peptides of interest into the cytosol in fact happens frequently enough, then i guess an exogenous antigen could be presented by MHC class I molecules!
Again Thank You very much!
1. There is a process that previously formed MHC-peptide complex can be exchanged or presented on the antigen presenting cell without going thru the exogenous nor endogenous antigen presentation called "cross dressing". The mechanism is still unclear but it is indeed occur.
2. By theory there shouldnt be any "free MHC I". As you mentioned MHC I can be expressed on surface only when a peptide is loaded on MHC I and coporated with Beta2 microglubulin.
3. The exgenous antigen can be uptake into the cell and form a endosome which will be fused with lysosome and procressed via exogenous antigen presentation. But in professional antigen presenting cell e.g. dendritic cell, there are some factor such as pH inside the endosome is not optimal for the enzyme so the endosome leaked and antigen get into cytosol subsequently processed like the cytosolic antigen instead. This process is called cross-presentation. It is an important property of professional APC in the cancer field. However, the full detail of the process is still inconclusive.
4. DCs is considered as the most efficient APC because a) DC expresses high levels of MHC and co-stimulatory molecules required for T cell activation b) DCs such as cDC1 posseses high cross-presentation activity (not every cell can do that).
I hope this answer might help you.
Cheers
Thanks Niphat Jirapongwattana
I am about to conduct a T Elispot for immunogenicity of MHC I peptides and am presented with the same challenge. I came across this paper: Article Cross-dressing: An alternative mechanism for antigen presentation
.However, cross-dressing appears to address transfer of entire MHC I - peptide complexes among immune cells, rather than the displacement of a pre-bound peptide by another (with the MHC I intact on the APC). Do you have a similar understanding or perhaps another source to support this?
My colleagues & I think the displacement of a pre-bound peptide by exogenous peptides may happen due to stoichiometry. If you artificially present a high concentration of exogenous peptide, it should be able to displace what is pre-bound particularly if the exogenous peptide is predicted to bind to MHC at high affinity? Just a thought.