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A few comments might be worth considering in response to Eric MIller and others
when attempting to relate CD69
expression to T cell activation:
First one should recognized that CD69 expression is NOT a replacement for 3H-
thymidine - although in some circumstances it may qualtitatively and/or
quantitatively parallel proliferative responses. CD69 expression is a measure
of activation in that it reflects the early requirements for cellular responses
to provocative stimuli - i.e. one can show antigen specificity, requirement for
antigen presentation, involvement of co-receptors (eg. CD28), and the nature of
stimulus. Furthermore we have recently shown using intracellular staining
techniques (to be published in August Blood) that T cells which express the
major T cell cytokines in response to various stimuli are all CD69 positive.
One should consider the rationale for measuring this response just as one should
consider the rationale for measuring the proliferative response - which can also
produce inherently misleading information - since it represents a later
consequence of cell activation, most of the active cytokine expression has
already occurred - with the potential to generate anomolous bystander
proliferative responses unrelated to the specific stimulus. I think what is
important here is not to relate CD69 expression to 3H-thymidine - but rather to
relate CD69 expression or proliferative responses or cytokine expression or etc.
to the clinical or biological problem one wishes to address. The value of CD69
expression is that it is rapid, provides cell subset information, and can be
performed in whole blood. We have already observed this relationship in some
early clinical studies we have performed - The hope is that by using a more
refined set of markers (e.g. cytokines) we will be able to provide additional
rationale for monitoring immune function using this approach.
Hope this helps - I would be interested to know what clinical question you are
trying to monitor. - Skip Maino
_______________________________________________________________________________
Subject: Re: lymph.function assay by CD69 and CD71.
From: Eric Miller <miller@icrf.icnet.uk> at INTERNET
Date: 7/3/95 12:54 PM
On Fri, 30 Jun 1995 brettT@qimr.edu.au wrote:
>
>
> We have just started using a CD69 and CD71 based lymphocyte function
> assay (based on some work I've previously done and the BDIS paper in Cytometry
> 20:127-133 (1995)).
> Generally I am seeking a group opinion on whether this method is valid
> to replace the tritiated Thymidine method, and, what ways can it be improved
> to make it more viable an alternative.
>
> The method uses ficolled lymphocytes (1million) stimulated for 4 hours
> with PWM, Con A, CD2, PHA, SEB, Candida A., PMA as a maximal control and an
> unstimulated control. These are then FACSed 4 hours using CD3/CD69 for
> activation and again at 16hours with CD3/CD71 for proliferation.
>
>
> My Questions:
>
> 1. Is there a need for an additional proliferation marker (eg. CD25) or is
> CD71 alone acceptable.
>
> 2. Would more co-stimulatory mitogens (eg. CD2 and 28) be more relevant
> clinically to test activation/function than mitogens like PWM and ConA.
>
> 3. Would DNA profiles be of any real advantage in this case.
My only comment on this would be that BrDU incorporation would probably
give the closest correlation with tritiated thymidine, and would also
give you a DNA profile to compare results with if you wished.
> Brett Teale
> Queensland Institute of Medical Research/Dept.of Immunology,
> Royal Brisbane Hospital
> Queensland
> Australia
>
> brettT@qimr.edu.au
Eric Miller, ICRF,Edinburgh UK
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