Summary T-cell stimulation with anti-CD3 and anti-CD28

Xincheng Zheng xchzheng at
Tue Oct 17 09:34:39 EDT 2006

Dear Anja and all:

Do you have any experience with monkey (cyno and rhesus) T cell stimulation
with anti-CD3 in vitro. For human PBMC, I can have very good stimulation
with OKT3 alone (from ebioscience, 0.1ug/ml in EHAA medium). But for monkey
T cells, I used SP34-2 clone (from BD at 0.1 or 0.3ug/ml, without CD28) but
never got good result. 

Can anybody help with this? Many thanks. 

Have a nice week.

Xincheng Zheng (Ted)

-----Original Message-----
From: Anja Scholzen [mailto:anja.scholzen at] 
Sent: Thursday, October 12, 2006 5:14 PM
To: cyto-inbox
Subject: Summary T-cell stimulation with anti-CD3 and anti-CD28

Dear all.

Thanks to everyone!
As I got quite a few requests, below are the answers I got.


You seem to be the only one on the list wanting to touch such a hot
potato as CD28 stimulation. However, you seem not to be the only one to
be puzzled about T-cell stimulation
( page37. You
might want to get hold of some TGN1412 as those guys seem to have
similar problems as you have.
OKT 3 is a clone from ortho biotech and is still available. Trying to
buy that antibody from BD is indeed an entertaining thought. Both clones
go for the epsilon chain of the T-cell receptor, UCHT1 is an IgG1 and
OKT3 is an IgG2a, but they should be used as Fab2's to avoid
interference from the 'other end'
>From the days of lymphocyte stimulation with purified material I
remember that - the better the purification the worse the cell
stimulation - as monocyte help was essential. Also crosslinking is
different depending on concentration and presentation (soluble vs
surface adhered).
See for example
Did you purify or did you try whole blood, fragments or whole antibodies.
Whilst we are just told that by my former employer big U that 'frozen is
good' I wouldn't apply it to those assays

When you want to stimulate the PBMC with CD3/CD28
antibody. it should work with my experience.

I used CD3 Cd28 antibodies from BD. With PBMC, I only
used soluble CD3(1 ug/ml) plus CD28(0.3ug/ml),
incubated PBMC for 8-24 hours and lyse the cells for
total RNA isolation(8h) or collect supernatant for

WHich concentrations did you used and how long have
you incubated cells with CD3 and CD28 antibody.

If you stimulate the purified CD4, you can not suceed
with soluble antibodies. You need to use either
pre-coated plates with CD3 antibody or you can direcly
use CD3/CD28 Dynalbeads.

OKT3 is definitely more reliable as a T cell mitogen and I agree that it
is difficult to source a commercial supply.  We bought the hybridoma
from ATCC and use the supernatant which works well even without
purification or concentration.	It works in solid- or liquid-phase in
our hands.


It works both immobilised (solid-phase) and in solution and we use it
with anti-CD28 (NALE from BD).


I used the combination of CD3
and CD28 to stimulate PBMNC's with good results. It is quit some time ago so
I don't
remember the details. We also used OKT3 with similar results (the OKT3 was a
left over
from an anti rejection therapy).


Haven't done it myself but a clinical fellow did it around the time I
joined the lab. Do you have some friendly clinicians doing transplant
work? OKT3 is used therapeutically and there is usually a little to
spare after injections are done.


I have had great success with the anti-CD3 clone
MEM-57 from abcam. I cross-link it with pharmingen's
goat antimouse Ig and get very consistent T cell


The problem with the two different clones is one of Fc recognition by
the donor, most likely.  If you "fix" the UCTH clone (solid phase) for
instance, you won't have a problem as it can cross link then.  You will
need accessory cells for the 2nd signal (or anti-CD28).


I'm not particularly experienced in this field, but I've use an invitrogen
product T-cell expander (Dynabeads coated with CD3/CD28).
It was fairly successful and easy to use.

*** I asked Miltenyi which anti-CD3 clone they use in this kit, but
unfortunately this information is not readily available (Anja)


Cheers, Anja

Anja Scholzen
PhD Student

Burnet Institute at Austin
Studley Road, Heidelberg 3084
VIC, Australia
Tel.:0061 3 9287 0673
Fax: 0061 3 9287 0600

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