We are co-culturing primary alveolar cells with PBMCs, the cells share the same environment but they are not in physical contact. Depending on the experiment number, PBMCs get activated and disrupt the epithelial cells after 24h, but this is something that does not always occur. We wonder if it is possible that our problem is a match in HLA considering that the epithelial and PBMCs are from different donors.
Is it possible that a HLA mismatch could be the cause of this? And if this is the case, which loci would you recommend to HLA type?
Thanks!
If the cells are separated by the transwell system (or some other system) and is never in direct cell to cell contact, I don't see how you can have a HLA-mismatch reaction.
The most obvious would be your PBMC donors. Or somehow during isolation you are activating your PBMCs, not a lot, just enough to start the process.
Unless somehow you are contiminating your samples with an exogenous source of stimulant?
Thank you very much for your answer.
There is some literature reporting that HLA can travel on exosomes, so that is why we considered if an HLA-mismatch would be a possibility. We obtained epithelial cells from different donors but in most experiments PBMC were from the same donor, we isolated the cells and kept them frozen until the day of the assay. In some cases we saw activation and in some others not.
Also, we compared the inflammatory state of the PBMCs (after thawing) either alone or in co-culture with the epithelial cells and we only saw activation in the presence of the epithelial cells. So maybe, epithelial cells release some cytokines/molecules depending on the patient that make the PBMC get activated.
It is indeed getting quite tricky to determine what is the cause of the activation.
Dear Nuria,
I did not know your cell co-culture system, but I am sure, that small molecules secreted from your PBMCs, like anti HLA antibodies (and complement molecules), could kill your primary alveolar cells.
Perhaps intensive washing of your PBMCs before the co-culture, could reduce your problem.
I would always use PBMCs and primary alveolar cells from the same donor!
If this is not possible, I would use leastwise HLA class I (HLA-A and B) matched PBMCs.
To give an example: HLA-A2, A1, A3 and A24 and HLA-B7 and B8 are frequently in the most human populations.
Best regards,
Thomas
Yes, that is true. but HLA mismatch is usually recognized by T cells. And I'm not even sure they are large enough to set off a T cell response. But exosomes by themselves can stimulate activation... although I think this mostly depends on what is contained in the exosomes.
What is the cytokine profile of these activated cultures?
Dear Thomas and Shen-An,
Thanks for your answers. We followed the activation by checking IL-8 in the supernatants, and the decrease in barrier function correlated with the increase in this cytokine. What is intriguing me is that if it is the thawing that activates them, then every time we used PBMCs alone they would've damaged the epithelial cells, and this is happening only in some experiments (which also correlates with different donors for the epithelial cells). Thanks for the suggestions for HLA-matching!
Hi Nuria,
The increasing levels of IL-8 that you are detecting makes me think that it's actually your epithelial cells that are being activated somehow. Did your healthy donors for the epithelial cells have some underlying/undetected "infections"?
Hope you are able to get to the bottom of this soon. Or you might just have accept the variance and split the groups, using only those cultures with lower levels of IL-8.