[Cytometry] Longevity of T cell activation markers HLA-DR and Ki-67

Howard Shapiro hms at shapirolab.com
Thu Feb 13 17:50:49 EST 2014

Calman Prussin wrote:

> We have identified a subpopulation of CD4 T cells in our eosinophilic GI disease patients that appear to be activated in vivo, in that they express increased HLA-DR, and Ki-67.  We'd like to culture the PBMC under "resting" conditions (no added cytokines) to see if these activation markers are down regulated. We can keep PBMC alive in vitro for 7 days under these conditions.
> Question: Does anyone know how long HLA-DR and Ki-67 stay up once the activation stimulus is removed?

I see that my recollections from 30 years ago that HLA-DR would drop off after 7 days have been confirmed.

I would once again like to beat the drum for replacing Ki-67 with other markers where appropriate, and lymphocyte activation seems to be an area where some multiparameter analysis that includes measurements of DNA and RNA  as well as surface and intracellular antigenic markers can provide a clue as to whether or not Ki-67 can be dispensed with.

In the late 1970s and early 1980s Darzynkiewicz et al used acridine orange to demonstrate increases in RNA and subsequently DNA in a 12-30 hr time frame after lymphocyte stimulation, and rhodamine 123 to show what was later recognized as an increase in mitochondrial membrane potential paralleling the known time course of RNA content elevation.

I introduced the combination of Hoechst 33342 and pyronin Y (Ho/PY) for DNA and RNA staining in 1981, based on Brachet’s demonstration in the 1930s and 1940s that absorption measurements of cells stained by methyl green and pyronin (a dye combination introduced for blood staining by Ehrlich around 1880) could provide quantification of DNA and RNA content previously available only using UV absorption measurement at 260 nm with and without RNAse treatment. Although one did need a UV source to excite the Hoechst dye (you can now usually get away with violet, and there are alternatives), enough RNA-bound pyronin is present even in quiescent cells for its fluorescence at ~580 nm to be measured with 488 nm excitation while simultaneously measuring immunofluorescence from fluorescein- and PE/Cy5-labeled antibodies. 

My colleagues and I examined lymphocyte activation combining Ho/PY labeling with staining with xantibodies to vsurface markers, and found that, in general, intensity of staining with anti-CD71 antibodies, which bind to cell surface receptors for transferrin, tracked RNA content as indicated by PY staining; quiescent (G0 or G1q) cells had low levels of both RNA and CD71, whereas elevated levels of both were present in G1, S, and G2/M phase cells. The staining pattern reported by others with Ki-67 is similar to what we have observed for RNA and CD71. Since I haven’t done anything with lymphocyte activation for decades, and never looked at Ki-67, I keep pestering other people to do definitive experiments using Ho/PY and fluorescent antibodies to both CD71 and Ki-67. The best reason for doing this is that if CD71 and/or RNA can provide the same information as to cell cycle phase for which Ki-67 is now used, one can stain living cells with Ho/PY and anti-CD71 and/or PY, enabling them to be sorted for further biological manipulation; it is also easier to do surface staining than it is to do intracellular staining.

Both RNA and CD71 levels in stimulated lymphocytes peak at 48-72 hr and drop markedly between 120 and 168 hr (the CD71 time course is reported in Biselli R, Matricardi PM, D'Amelio R, Fattorossi A. Multiparametric flow cytometric analysis of the kinetics of surface molecule expression after polyclonal activation of human peripheral blood T lymphocytes. Scand J Immunol. 1992 Apr;35(4):439-47. PubMed PMID:  373002). I’m betting that Ki-67 will show a similar time course. So might mitochondrial membrane potential, but that’s another story.

I may actually be doing some lymphocyte activation experiments within the next year or so in conjunction with testing one of the cheap imaging cytometers about which I keep talking, but malaria remains the priority application for those. It would be much better to have somebody still actively working on lymphocytes to do the experiments. I’m OK at cytometry, but cells don’t like me much.


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