I have a compound that inhibits MHC class II antigen presentation of OVA protein but not of OVA peptide by peritoneal macrophages, does this mean that it inhibits the antigen processing or degradation of CLIP and/or loading of peptide on to MHC or both? The compound has no effect on presentation of OVA peptide when added to fixed or unfixed macrophages.
Thank you,
We can not say exactly. Peptide presentation works by default by binding to the MHC II on the surface. But the presentation of OVA requires its uptake, processing, and loading. You need to have proper controls to decipher this. For example, if you use OVA-Alexa 488, you can test whether OVA is uptaken and if you use DQ-OVA, you can test whether the OVA is processed in the cell or not. The question of loading problems can be asked. If the cell is dead or intracellular transport is inhibited by your compound, the OVA can not be processed and presented. Even MHC II turnover by the cell also affected by some compounds which again affect your antigen presentation. It is quite complex considering the number of steps and factors involved in the presentation of antigen by the antigen presenting cells.
Just check more published literature about the compound you are using and make some initial hypotheses about what it can be related to the antigen processing and presentation.
Hi,
@Ramachandramouli Budida,
I have checked for antigen uptake, and the compound does not affect antigen uptake
Hi Komal,
Your speculations may stand true, but before that confirm whether the effect of your compound is not specific only for ova protein. In that connection, you may use another protein/s (other than ova) to check whether your compound have a more generalized effect. After establishing that you can go ahead more systematically to address raised possibilities.
Hope this may be helpful.
@ Komal,
Then you can check whether antigen processing is inhibited. If it is a question of loading, you can also check for MHCII-OVA complex on the cell surface by an antibody specific for this complex. Did you check the cell viability when you treat with this compound? And you can also check for MHCII turnover by doing a time point analysis for MHCII surface expression. In general, the MHCII surface expression goes up with time in vitro. If this is inhibited by your compound, it is not loading but the turnover of MHCII is inhibited.
Good luck
@Ramachandramouli Budida,
It has been observed that the compound does not affect surface expression of MHC II or costimulatory molecules on the surface of macrophages. It is also found that the compound inhibits MHCII-OVA complex on the cell surface. the compound does not cell viability.
@Varun Shah,
the compound also inhibits antigen presentation of other antigens.
Thank you,
Then your results suggest inhibition of processing or loading (excluding effects on MHCII turnover and intracellular transport). How did you check the MHCII-OVA complex on the surface? and How did you check the uptake of OVA by the macrophages?
Hello,
@Pedro A Reche,
I had used MHC II specific peptide, so it is presented by MHC II
Hi,
@Ramachandramouli Budida ,
I had used FITC OVA to check antigen uptake by macrophages.
Komal, it seems your compound inhibits antigen degradation. It is unclear however whether antigen loading will occur in the cell surface or within mhcii vesicles that fuse with ova endocytic particles. Pptide loading on the cell surface will mostly occur if empty mhcii molecules can reach the cell surface. Empty mhci molecules can reach the cell surface but i do not know about mhcii molecules. Here goes a paper of many on the subject
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3839614/