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>>>"Unfortunately this stimulatory condition leads to IFN-gamma
>>>production even
in Th2-like cells"
Please give more details. Both Oppenshaw/O'Garra (mouse) as well as
Bernhardt Hemmer (human) here at the NIH have looked at parallel
experiments in defined T cell clones and shown that antigen and PMA/iono
give the same results in terms of dominant Th1 or Th2 profiles. There
was no evidence of IFN production in Th2 clones.
In short term (1-3 weeks) human Th2 lines that we have made, we see no
IFN production after PMA/iono.
In fresh ex vivo T cells we have demonstrated (JI, 154, p. 4294) that
there is very only moderate coexpression of IL-4 and IFN and essentially
no coexpression of IL-5 and IFN. The vast majority of IL-4 producers do
NOT coexpress IFN.
>>>we failed to detect any
>>>IL-4, or IL-5 despite the fact that we detect high levels of IL-5
>>>secretion
>>under these conditions in our generated Th2-like cells.
We have done parallel ELISA and cytokine flow experiments and found good
correlation between the two for IL-4, IL-5 and IFN (same JI). IL-4 can
be difficult to detect even with the new directly conjugated reagents.
You may wish to use an eosinophilic subject as a control.
>> The bottom line is that we are still struggling with this technique
Unfortunately, the Th2 cytokines are more difficult than IL-2 and IFN.
There certainly is a steep learning curve for the technique.
>----------
>From: Julie Auger
>Sent: Thursday, May 30, 1996 7:28PM
>To: Cytometry Mailing List
>Subject: Re: intracellular IL-4 and IFN-gamma
>
>
>>Date: Thu, 30 May 1996 10:38:00 -0500
>>X-Sender: gvsevent@flowcity.bsd.uchicago.edu
>>To: Julie Auger <jauger@flowcity.bsd.uchicago.edu>
>>From: gvsevent@flowcity.bsd.uchicago.edu (Gijs A. van Seventer, Ph.D.)
>>Subject: Re:
>>
>>We have performed intracellular staining for cytokines with reagents from
>>Pharmingen. We tried various ways of stimulating T cells and the best one
>>(and recommended by pharmingen) is stimulation with PMA/ionomycin.
>>Unfortunately this stimulatory condition leads to IFN-gamma production even
>>in Th2-like cells. Great intercellular staining was observed with this
>>activation methods for IFN-gamma, IL-2, TNF-alpha, we failed to detect any
>>IL-4, or IL-5 despite the fact that we detect high levels of IL-5 secretion
>>under these conditions in our generated Th2-like cells. The bottom line is
>>that we are still struggling with this technique and have not found a
>>solution to measure IFN-gamma vs. e.g Il-5 in primary cells even after 1 ot
>>2 stimulations in vitro. Moreover the most promising activation condition
>>induces IFN-gamma in Th2 cells that would not secrete IFN-gamma when
>>stimulated through the TCR/CD3 complex. Succes further.
>>Gijs van Seventer
>>
>>
>>
>>>>X-Sender: brian.rabinovich@mailbox16.utcc.utoronto.ca
>>>>Date: Tue, 28 May 1996 03:37:53 -0400
>>>>To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
>>>>From: brian.rabinovich@utoronto.ca (Brian Rabinovich)
>>>>
>>>>
>>>>Addressed to all those who may be able to aid in the process of staining
>>>>for intracellular cytokines using fluorescently tagged mAbs for FACS
>>>>analysis on peripheral blood:
>>>>
>>>>I am currently performing an assay involving intracellular staining of
>>>>cytokines from peripheral human blood to assess TH1 and TH2 populations
>>>>of CD4+ cells (The protocol I am using is below) . I am currently NOT
>>>>stimulating the blood after obtaing it as we wish to assess any possible
>>>>alterations in TH1:TH2 between controls and patients with vascular disease
>>>>in as pure a fashion as possible. I am finding, however, that unstimulated
>>>>CD4+ cells from peripheral blood stain very poorly for both IL-4 and
>>>>IFN-gamma, in most cases. I was hoping that someone might have a simple
>>>>protocol for stimulating CD4+ cells to synthesize IL-4 or IFN-gamma
>>>>depending on their phenotype so that I can stain for a proportionally
>>>>amplified cytokine profile. Also, because we are drawing blood from
>>>>patients and controls on a regular basis, I am looking for a stimulation
>>>>procedure which is as simple and least time consuming as possible.
>>>>Please
>>>>help.....Brian A. Rabinovich, Research Institute, The Wellesley Hospital,
>>>>University of Toronto. E MAIL: brian.rabinovich@utoronto.ca
>>>>
>>>>P.S. Please forward this mesage to anyone else you think could lend
>>>>a hand
>>>>
>>>
>>>
>>>
>>
>>
>>----------------------------------------------------------------------------
>>---------
>>Gijs van Seventer, Ph.D.
>>Assistant Professor
>>Department of Pathology
>>Committee on Immunology
>>University of Chicago
>>5841 S. Maryland Avenue
>>SBRI, J-541A, MC 1089
>>Chicago, IL 60637-1463
>>Tel:312-702-9997
>>Fax:312-702-3701
>>
>>
>>
>
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