I'm evaluating the capacity of some epithelial cell supernatants to induce dendritic cell maturation. I take murine bone-marrow-derived dendritic cells on day 6 (DCs) and culture them for 48 h in the presence of culture medium alone (immature DCs), 1 ug/ml LPS (mature DCs) and my supernatants at 25% concentration. I observe a comparable increase in MHC II and CD86 expression in all treatments but in immature DCs, as expected. I then take the DCs, wash them twice and culture them again at a 1:10 ratio with CFSE-labelled allogeneic T cells. I observe significant proliferation with SN-exposed DCs on day 3 (up to 4-5 rounds of proliferation) and not with LPS (only 2-3 round) at this timepoint. If I wait up to day 5, LPS DCs induce proliferation similar to that observed for the SNs on day 3, and at this point, the SN DC culture have considerable cell death. I cannot conclude from these experiments that SN DCs are more efficient at T cell stimulation, can I? Do you have any suggestions? What do you think is going on in my experimental setup?
I ran my experiments in parallel and at least 4 times, with different batches of epithelial SNs. These results are very reproducible and I had good viability on day 3, so I guess that they are reliable as well. LPS induced the highest MHC II expression in DCs, and some SNs induced more MHC II expression than others, but none to the same level than LPS. CD86 expression was not as reproducible, and some SNs induced higher expression than LPS Regardless of MHC II & CD86 expression levels, all SNs invoked the same degree of DC-induced T cell proliferation. I'm afraid I might be missing something here. I had always thought that LPS was one of the strongest stimuli for DCs.
you might want to separate your T cell populations. Removing Tregs (CD25+) cells for example, could give cleaner results.
it looks like SN is quick to stimulate T cells whereas LPS DCs do it through a different pathway. May be different molecules are involved in the stimulation.Increased cell death means SN has some effect on t cell aging or programming or nutrient limitation etc!
It may be, observed effects are due to IL-12, which can be presented in LPS stimulated cultures. In some settings, this cytokine can partially suppress T cell proliferation, but stimulate formation of CTLs. To understand full picture, you will need to see on the phenotype and function of cultured T cells. Then, you will see possible explanations and ways to study the effects on cytokine level. Good luck!
hi Jeremias
just curious about why you used 25% concentration. Is your effect stll there if you dilute this? I mean, are you saturating DCs with -otherwise- an obvious stimulation?.
Is possible that your conditioned media is contaminated with LPS or any other PAMP?
More than 5d in a CFSE assay means a lot of cell death. I would try diluting the conditioned media and see how it works
Best
Dear Francesc,
yours is a great suggestion! Many published reports use 25% concentration for assaying epithelial supernatants, and sometimes more. I simply started with this concentration and I obviously observed considerable effects. My main worry is that I might be missing something such as culture contamination. There is no evident contamination in my epithelial cell cultures and mycoplasma tests are negative. I will try more dilute concentrations of my SNs, perhaps it is a saturation issue.
Thank you!
Antonio Riva has raised some valid points on the repetition and the reproducibility of the experiments. If you are confident on the reproducibility, then discuss on your results, which may be right. lalitha kabilan
The kinetics of allogeneic T cell stimulation has been a topic of controversy since the mid 1970's. Stellar Knight and Johnathan Howard have both authored lengthy papers on the subject.
The first issue ti deal with is the purity of your DC's. A B cell contamination can realy skew your LPS results. Second is the 1:10 ratio which seems on the low side to me. Traditionally these assays are performed with 3 or 4 different ratios. This should give you a better idea of what is going on and what is going wrong.
Hope this helps.
DW
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