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Interference by LPS in GM-CSF ELISPOT

Guest Sara

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I have recently started analyzing T cell activation with GM-CSF ELISPOT. PBMCs are stimulated with various antigens and my read-out is GM-CSF as well as IL-2, IFN-g and IL-17A. I am concerned that I may get false positives, especially in the GM-CSF assay, due to LPS-contamination of my antigens (I get a lot of spots) and that the signal may originate from monocytes and not T cells. Do you have any experience of this and/or any advice on how to avoid this problem. 



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Dear Sara, welcome to the Forum!



I think your reasoning on this issue is absolutely correct. Monocytes are very cytokine-capable cells and an inherent part of all PBMCs preparations (approximately 10-20% of PBMC). In cases where antigens are contaminated by LPS (or any PAMP for that matter) these monocytes will get activated and will secrete a tremendous amount of both inflammatory and anti-inflammatory cytokines during the 24-72h incubation that you typically do during an ELISpot experiment. Now, monocytes are, at least to my knowledge, not able to produce IFN-g and IL-17A in response to stimuli, but they do in fact secrete GM-CSF. That is, your concern of getting false positives is in this case certainly merited. 


At Mabtech we often use an LPS inhibitor called Polymyxin B when examining new antigens. This compound is known for binding to the lipid A moiety of the LPS molecule and inhibiting its activity. I would therefore suggest setting up the following experiment in your GM-CSF ELISpot:


1. Cells only

2. Cells + antigen 

3. Cells + antigen+ PolymyxinB

4. Cells + PolymyxinB



If the end result is that in "3" the GM-CSF spots goes down significantly compared to "2" (or totally disappear), well, that is then a very strong indication that these spots do in fact originate from contamining LPS, and not from antigen specific T-cells. If the spots are not influenced, a contamination could still be the orginial source as Polymyxin B only inhibits LPS, and not other PAMPs such as flagellin, viral RNA, viral DNA and so on. 



To test your antigens against a much broader spectrum of PAMP contaminants, we at Mabtech are very fond of setting up something we call "monocyte ELISpot" or "monocyte FluoroSpot". By incubating the antigens in an IL-6 ELISpot/FluoroSpot with a much lower number of PBMCs than normal (around 2,500-10,000 cells/well), you are able to screen for all sorts of contaminating PAMPs by looking at the induced number of IL-6 spots as compared to medium ctrl. If your antigens contain anything that will stimulate the 250-2000 monocytes in these ELISpot wells you will see an increase in the number of IL-6 secreting monocytes and know that something "is going on". The benefit here is that the ELISpot assay is so sensitive and that your are actually looking at living, cultured monocytes themselves, directly exvivo. IL-6 is chosen since its background tend to be rather low at these cell concentrations and its secretion is strongly induced by most pattern-recognition receptor pathways. Any possible contaminating PAMP can be detected with this setup, even such we don't even know exist yet!


People from R&D at Mabtech has just recently published an article in an open journal called Cells where they used this strategy for looking at the IL-6/IL-1beta secretion by monocytes in FluoroSpot using three different antigens: C.albicans (CC), Tetanus (TT) and PPD. 





Strikingly, in this experiment, PPD induced the monocytes to secrete both IL-6 and IL-1beta at quite high frequencies, whereas TT and CC did not. Obviously, the PPD preparation contains something that is making the monocytes activated and secreting these inflammatory cytokines. The low cell number more or less guarantees that we are looking at a monocyte-derived cytokines and nothing else.



Here is a link to another free methodological publication detailing the use and setup of monocyte FluoroSpot using Mabtech kits:







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