Re: proliferating lymphocytes in peripheral blood and CD25 expression

Petros Christopoulos wrote:

>I would like to ask if anybody knows about the relationship of 
>expression of classical
>proliferation markers (like Ki-67 or the transferrin receptor) and 
>CD25 (which is also an activation marker) in peripheral blood lymphocytes.
>Do they give similar results, do they correlate or are they totally 
>independent of each
>other ?

Time to start another winning streak for Mailing List submissions...

See Figure 10-7, p.460, in the 4th Edition of Practical Flow 
Cytometry (available without charge on the Invitrogen/Molecular 
Probes web site, http://probes.invitrogen.com/) for plots relating 
transferrin receptor (CD71) expression and RNA content to DNA 
content. I have speculated in the book and on this Mailing List that 
Ki-67 and CD71 should correlate almost absolutely in proliferating 
lymphocytes and probably in most or all other cell types, but, when I 
just looked on PubMed, I could not find any publication suggesting 
that both had been measured simultaneously in the same cells at the 
same time. This really needs to be done; there have been over 100 
papers published referring to Ki-67 in the past year, representing a 
lot of work by many people. Detecting Ki-67 requires permeabilization 
and is only possible in fixed cells, whereas detecting CD71 requires 
only surface staining, meaning that live cells can easily be analyzed 
and sorted based on CD71 status, but not based on Ki-67 status. If 
the two markers are as well correlated as I suspect, a switch from 
Ki-67 to CD71 staining will save both time and money, and, more 
importantly, allow researchers to do further functional studies on 
live cells precisely characterized as to cell cycle position. Such 
studies can, in at least some cases (see the work of Edward Srour et 
al), be done using Hoechst 33342/Pyronin Y staining for DNA and RNA, 
but pyronin Y can be toxic.


[]


The above, unpublished data from experiments done eight or nine years 
ago, shows that in CD4+ lymphocytes gated from cells in a mixed 
lymphocyte reaction, CD25 expression apparently occurs all cells in 
G1, S, G2, and M phases of the cell cycle (the quadrant lines are not 
set well; the horizontal line should be lower, and the vertical line 
closer to the vertical axis). We only did simultaneous staining of 
CD25 and CD71 on a couple of occasions, since we were typically 
staining for DNA (Hoechst 33342), RNA (pyronin Y), and CD4 or CD8 
(PE-Cy5) as well as for the activation antigen (fluorescein). I could 
not find a plot of CD25 vs. CD71, but my recollection is that they 
were close to absolutely correlated, as the plot above and the figure 
in the book would suggest.

>If they do correlate, how would an increased activation status of 
>the immune system
>(increased percentage or number of HLA-DR+ cells) be expected to 
>influence that
>relationship ?

Again, based on work done many years ago, we found that HLA-DR 
expression was a rather poor indicator of lymphocyte activation; 
HLA-DR did not come up until about 72 hr after cells were stimulated 
by lectin or alloantigen, and HLA-DR was not expressed on many cells 
that could be shown to be in proliferative (G1,S, G2, M) phases of the cycle.

In the era of 18-color flow cytometric analysis, it should not be 
that difficult to answer your question definitively by simultaneously 
measuring and subsequently correlating correlating expression of 
multiple activation antigens with DNA and RNA content (cell cycle 
phase) and with both T-cell phenotype (CD4+, CD8+, etc.) and memory 
class. That's more Mario Roederer's line of work than mine these days.

Holiday Greetings to all,

-Howard



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