Re: Lymphocyte Activation Markers
Howard Shapiro (hms@shapirolab.com)
Sat, 8 Jul 1995 22:50:06 -0400
Lymphocyte proliferation is, under normal circumstances, the consequence of
lymphocyte activation; measures of proliferation in response to mitogen or
antigen stimulation have, accordingly, been employed as indicators of
cellular immune responsiveness. The method most widely used is detection of
thymidine incorporation into DNA, although direct observation by time lapse
photography was done as well in the early days. By the mid-1970's,
Braunsteiner, Darzynkiewicz, and others had demonstrated that measurements
of DNA and RNA content correlated well with proliferation as determinedby
thymidine incorporation; however, while DNA synthesis and cell division
require a lag period of about 30 hr, RNA synthesis occurs earlier, at around
20 hr. By the late 1970's, people became interested in earlier indicators
of lymphocyte activation, including changes in membrane potential, calcium
content, pH, and cytoplasmic structure. In the early 1980's, it became
possible to detect a variety of antigens newly expressed or increased in
quantity on the surface of activated lymphocytes; in the case of T-cells,
these include what are now classified as CD25 (IL-2 receptor), CD69, CD71
(transferrin receptor), CD98 (formerly known as 4F2), and HLA-DR.
Multiparameter studies examining two or more such antigens in phenotypically
defined subpopulations are few in number, largely because four or more
colors are required. Similarly, few if any activation antigens have been
correlated with RNA content (which cannot be done readily using the more
common acridine orange staining method but can using pyronin Y). CD69
appears early, but is known to disappear after about 48 hr; Skip Maino's
posting should have more details on this. Old work my colleagues and I did
showed good correlation between CD25 or CD98 and thymidine uptake; I am
starting to do another round of such work. More recently, it has been shown
that proliferative fraction in lymphocytes can be estimated by mathematical
analysis of fluorescence of tracking dyes in lymphocyte membranes. It is
known that a number of hurdles must be jumped between calcium influx a few
minutes after stimulation and DNA synthesis; there may also be a hurdle or
two between CD69 expression and DNA synthesis. If you wanted to correlate
antigen display with proliferation, you would probably therefore be best off
with antigens which persist during the entire proliferative phase. I hope
that clarifies things.
--Howard Shapiro
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