Bunny, you are in a bad situation no matter what. Besides the issue
of trying to identify CD8 T cells from NK (you should set the markers
at the high population), you are gating RA+ vs RA- cells. As many
people have demonstrated quite extensively over the years, these are
highly heterogeneous populations. CD8+ RA+ are a mixture of naive
and memory, with VERY different functionality. So any results you
get on the RA+ population are going to principally driven by the
representation of naive & memory cells within that gate, which varies
strongly from individual to individual, and even more strongly when
considering pathogenic states like HIV, chemo-treated adults, etc.
Bottom line, the results you get on this gate will not be
particularly meaningful biologically.
In general, one should not use CD45RA by itself to look at RA+ T
cells (nor CD45RO by itself in or in combination with RA to look at
RO-/RA+ T cells). It's kind of like asking what fraction of
MHC-Class I-expressing cells secrete antibody (answer: it depends
solely on the proportion of B cells within the gate).
If you're limited to 3 color analysis, then there is no solution.
Unfortunately, it is IMPOSSIBLE to accurately identify naive T cells
using 2 color immunophenotyping (see De Rosa et al., Nat. Med., Feb
2001).
mr
>friends-
>
>I have a dilemma. I'm participating in a study analyzing
>chemokine receptors in MS patients in clinical trials. The
>combinations used are: CD4+/CD45RA+/CHEMOKINE and
>CD8+/CD45RA+/CHEMOKINE.
>
>Here is my question: for measuring the CD8/45RA portion, do
>I set the markers on background (isotype or
>unstained,whatever- that's not my current question) and
>accept ALL CD8+ (DIM AND BRIGHT), or specifically CD8 bright+/CD45RA+.
>
>For most of the chemokines, the results are the same (for
>either /CD8+ bright/45RA+ or CD8+ dim/45RA+). But for one
>marker the results are dramatically different.
>
>{I do understand that the CD8 dims's usually contain NK
>cells. I searched both cytometry archives and pubmed, and
>found many valuable discussions of the roles of these
>various subpopulations. For this study, adding more/or
>changing CD markers is out of the question.}
>
>So my main questions here are:
> 1)With the combinations described above, WHERE do people
>normally set the marker for CD8+ cells;
> and
>2) do you then report the data as "%chemokine receptor
>expression in activated CD8+45RA+ cells" ? or CD8+bright/CD45RA+
>
>--
>Bunny Cotleur
>Cleveland Clinic Foundation
>Neurosciences NC30
>9500 Euclid Avenue
>
>Cleveland, OH 44195
>
>(216) 444-1164
>cotleua@ccf.org
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