>Some of you may be doing intracellular staining for cytokines. I am >particularly interested in staining mouse lymphocytes under conditions as >close to ex vivo as possible. I am comparing cytokine responses of normal >and infected mice. My experience with PMA and ionomycin plus brefeldin A was >that relatively higher frequencies of cytokine-producing cells could be >obtained, but the difference between normal and infected mice seemed >obscured. I tried Ag and anti-CD28 plus brefeldin A as in vitro activation, >the frequency of positively stained cells was below 1% and such a number was >hard to reproduce. > >Wish to hear from you, and we can discuss more details. Thanks in advance. > >Hai Qi >Dept. of Pathology, UTMB Hai Qi, We have been struggling with very similar issues with respect to detection of intracellular cytokines ex vivo. In our case we often compare T cells primed in the presence vs. the absence of costimulation. As you mentioned, boosting ex vivo with PMA and Iono give good frequencies of cytokine-producing cells, but this is whether the mouse is immunized or not. Essentially, what we are both seeing is a subset of the normally occuring primed/memory population which has been developed in response to environmental antigens. In TCR transgenic models, where one can identify the antigen-specific cells using an anti-clonotypic antibody, P & I also show very good frequencies of cytokine-producing cells, but in our hands, this form of signaling in which the surface receptors are bypassed overcomes any costimulatory requirement during the priming step. I.e., cells primed in the absence of costimulation show the same frequency of cytokine-producers (using P&I) as cells primed under good costimulatory environments. This is not the case if cytokine production is measured by ELISA in response to restimulation with specific antigen (similar to your infected vs. uninfected). There are a few ways that we have overcome (to varying degrees) these problems: If analyzing a polyclonal population of T cells, we have found that plating cells ex vivo on plate-bound anti-CD3 and anti-CD28 (10ug/ml for coating) for 5 hours in monensin gives respectable frequencies of cytokine-producing cells (not as high as P&I, but this has the advantage of signaling via receptor ligation), and the production of cytokine during this restimulation period is dependent on good costimulation during primary stimulation. The best results we've had is in a TCR transgenic system where we can both identify the antigen-specific population and restimulate with soluble, specific <peptide> plus anti-CD28. We find that peptide+CD28 is actually superior to PMA&Iono in cytokine-producer frequency, and the expected costimulatory requirements are maintained. In your case, if you don't have a defined peptide (and/or clonotypic population) to work with, whole antigen may work; however, adding it during the 5 hour boost period won't, because there is not enough time for a significant amount of epitopes to be processed and presented to the primed T cells. In this case, we have had some <limited> success by pulsing LPS-primed spleen cells overnight with antigen, and using these as stimulators for the 5-hour ex vivo restim. Purified dendritic cells should work better. As a final comment, I (as I'm sure many others) would suggest that PMA and Ionomycin restimulation is useful as a test for whether a T cell has developed the <potential> to produce cytokine(s) at a most basic level (i.e. whether a cytokine gene locus is 'open' or 'closed'?), whereas receptor-mediated ligation using antibodies or antigen/MHC would reveal additional layers of signals which regulate the ultimate decision for cytokine production. Good luck and hope this helps, AW Andrew D. Wells, Ph.D. University of Pennsylvania Department of Medicine 904 Stellar-Chance Laboratories 422 Curie Boulevard Philadelphia, PA 19104 (215) 898-1951 (215) 573-2880 (FAX) adwells@mail.med.upenn.edu
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