Thankyou to all those who replied to my question on 'which activation marker for
leukocytes' , most appreciated.
Sorry if I have missed anyone.
For the benefit of others here are some of the responses that I received
Having studied activation markers for a number of years in mice and now
nonhuman primates, I have never been that impressed with my own findings
regarding CD25 or CD69 as markers of activation. Too often I have seen
little or no expression of either marker. Not that I doubt what is reported
in the literature, but CD69 is known to be only expressed transiently and
CD25 comes up very late (or not at all). One marker we have used very
successfully is CD44 (also called Pgp-1 or Ly24 in mice). Naive T cells are
always CD44 low, activated T cells and memory T cells are CD44 high.
I know that others have used combinations of CD45RA and CD45RO as markers of
activation in humans but that does not apply for mice as mAb for CD45RO do
not exist in mice.
For the record:
CD69, also called early activation marker - comes up early but is expressed
only transiently.
CD25 - IL2 receptor alpha chain. Comes up late in activation (or not at all)
CD44 - Ly24, Pgp-1. Adhesion molecule. Comes up later than CD69 but still
early (within first division by CFSE staining); maintained at high levels
afterwards, even into memory phase.
CD71 - I have no knowledge; can't say whether it would work or not or when
it's expressed.
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My experience is with human T-lymphocytes stimulated with PHA in fetal calf
serum-free medium.
At one hour after stimulation we can detect CD69 (17%-59% possitive cells)
and increase at 6 h (95-82%).
At 6 h, we can t detect CD25, but at 12 h the expression is possitive in all
donors tested (26-66% possitive cells) J leukocyte Biol (2001), in press.
I din't use CD71, but it is described that is expresed between CD69 and
CD25.
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We've spent the last year attempting to detect cytokine production
by activated T cells in primary and secondary MLRs. In summary:
We haven't used CD71 although I'm aware it is found on activated T-
& B-cells, macrophages and proliferating cells.
CD25 is the Il-2 receptor. We've have detected it in primary and
secondary MLRs.
CD69 is an early activation marker found on T cells. It is also
found on activated B cells, activated macrophages and activated NK cells.
The shortest time we have waited prior to adding Bref A/Monensin is
30 minutes. CD25 was detected, but CD69 expression was very weak. We now
wait 60 minutes prior to adding a protein secretion inhibitor - we have
found reasonable levels of CD69 expression after 1 hour. Levels seem to
begin dropping off after 24 hours, and after 48 hours, very little, if any
is detected.
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Kind of depends on the activation, but in general CD69 is up in 4-8 hours.
CD25 takes 12-24 to sometimes 48 hours to really come up. Don't know about
CD71
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I suggest that you use antibodies to detect CD71 for isolating activated T cells.
Since it is the transferrin receptor responsible for bringing iron into activated and
proliferating cells, it becomes upregulated and expressed on the surface of leukocytes
(T cells, B cells, and macrophages) as they leave G0 into G1.
Whereas CD69 becomes expressed on the same cell types during activation, it
becomes progressively downregulated through successive divisions. In contrast, CD71
expression remains elevated during proliferation. Although CD25 measures expression of
a unit of the IL-2 receptor that generally correlates with activation of
IL-2-responsive cells, its expression is variable depending on the subcategory and
differentiation status of the cell type.
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Heather, all these markers are activation markers that arise at
different time points...CD69 may be positive as early as 6 hrs with
CD25 following later and CD71 even after that. Typically people use
CD69 for induced cytokines as it coincides more with the production.
When you look for the cytokine depends upon your activation system,
the cells you are using, and what cytokine you are looking for..need
a few more details.
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CD69 is by definition the earliest expression marker.(10-45 min.) but doesn't stay
around long. CD25:IL-2 receptor expression comes in (I think) 1-5 hrs. and CD71:
Transferrin receptor stays around the longest.
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You can likely use antibody to any of the three markers you mention.
CD25 comprises part of the IL-2 receptor and was known in the earlier
literature as Tac (for T cell activation). CD71 is the transferrin
receptor and CD69 is an "activation" antigen that seems to be getting
murkier, although it has been widely used to detect stimulated cells.
I did detailed time course studies with anti-CD25 many years ago.
Peripheral blood lymphocytes normally have very low levels of CD25
expressed. Stimulation in vitro with polyclonal activators such as PHA
or anti-CD3 causes readily detectable amounts to begin appearing after
9-12 hours. By 24 hrs with these kinds of stimulation, both CD25 and
CD71 can be detected in high amounts on activated T cells. I think
anti-human CD25 and CD71 are available from many suppliers with several
different fluorochromes. The expression of these markers on activated
cells is not at all subtle, so there should be little trouble detecting
them.
I am sure you will get some good responses concerning anti-CD69 from
others who have more experience with it.
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