reproducibility of CD3 zeta p23/p21 ratio

TIM tim at besancon.net
Fri Jun 5 02:35:42 EST 1998


Hi,

I would like to use the CD3 zeta phosphorylation level as a way to
evaluate the ability of human naive T cells to be activated. The goal is
to follow up the immunosuppression of patients before and after
different types of treatments: if T cells are anerguzed, they should not
be able to phosphorylate optimally the CD3 zeta chain.

The method is the following: PBMC are stained with OKT3 (10g/ml, 4¡C,
20 min), washed with saline, incubated with azide-free GaM-Ig (10g/ml,
4¡C, 20 min). The cells are then pelleted and resuspended in RPMI 1640
at 37¡C for 10 min before lysing. Spined cell lysate are then separated
onto a 15% SDS-PAGE, blotted, revealed with 4G10 (anti-phospho-tyrosine)
+ peroxidase-conjugated GaMIg + ECL. The peak ratio of p23 versus p21 is
then determined by scanning the film.

I have a problem of reproducibility: although induction of p23 is very
good with normal donors (undetectable in unactivated cell lysates), the
p23/p21 ratio is variable from one experiment to another (ex: 0.3 to 0.5
in one experiment, 0.8 to 1.2 in a second one). The variability seems
more to be activation experiment-dependent than western-blot
experiment-dependent (two aliquots of the same cell lysates give
identical p23/p21 ratio in two independent WB). One people working in
another lab told me that she had also these problems when using fresh T
cells: one normal donor tested 3 times gave p23/p21 ratio values varying
from a factor 1 to 5.

Do you know how to reduce this activation experiment-dependent
variability?

Any informations about the relationship between CD3 zeta phosphorylation
and anergy when using naive T cells instead of T cell lines or clones?

What positive control of anergy do you suggest? (I would like to avoid
using T cell clones or lines: these cells are more homogenous than PBMC
and they phosphatase activity seems to be different). I have used 12
days-activated T cells (OKT3 100 ng/ml at d0, IL-2 500 u/ml from d3 to
d12) without any significant change in the p23/p21 ratio, as compared
with fresh PBMC, although they are far less alloreactive...

Many thanks for your reply

Eric ROBINET



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