RE: memory/naive CD8 and CD4 T cells

hi Mario!

can you and others weigh in on how to correctly identify mouse memory T cell subsets ?

thanks,
Rachel

=======================================================
Rachel M. Gerstein, Ph.D.
Assistant Professor
Department of Molecular Genetics and Microbiology
Graduate Program in Immunology/Virology
University of Massachusetts Medical School
55 Lake Avenue North
Worcester, MA 01655-0002
(508) 856-1044
(508) 856-5920 (FAX)


> ----------
> From: 	Mario Roederer
> Sent: 	Monday, March 7, 2005 4:54 PM
> To:	Cytometry Mailing List
> Subject:	Re: memory/naive CD8 and CD4 T cells
> 
> Please refer to multiple published manuscripts on this topic, plus 
> old email threads (dating in the mid to late 1990's) on the cytometry 
> list.
> 
> Basically, you CANNOT distinguish naive and memory solely on the 
> basis of CD45 isoforms.  There are memory cells that are 
> CD45RA+CD45RO- (like naive), and these can become very prevalent in 
> certain conditions (e.g., AIDS, chemotherapy, mucosal tissue 
> specimens).
> 
> You MUST use a second marker with CD45RA (or CD45RO), such as CD28, 
> CD27, CCR7, CD62L, CD95, CD57, or CD11a.
> 
> Our Nature Medicine paper from 2001 also shows that you really ought 
> to use at least 3 markers to uniquely identify naive T cells; using 
> "only" two gets you about 95-97% pure naive but 3 will get you 
> to >99% pure.
> 
> So, unfortunately, if your panel is as you describe, you cannot 
> adequately enumerate naive and memory T cells.
> 
> mr
> 
> At 4:54 PM +0100 3/5/05, Corrado Cilio wrote:
> >Hi all,
> >
> >I have a discussion going on in the lab concerning the analysis of 
> >memory/naive T cells by flow. We stain human peripheral lymphocytes 
> >with aCD3-FITC, aCD45RA-PE, aCD4- or aCD8-PerC and aCD45RO-APC in 
> >whole blood to determine the memory/naive pheotypes. How people do 
> >the analysis? We have three alternatives on the discussion table and 
> >would like to know your opinion, more to think?
> >
> >A. 1st gate: lymphogate SSC,FSC; 2nd gate CD3+CD4+ or CD8+; 3rd 
> >dotplot CD45RA vs CD45RO with quadrant to get the % of different 
> >populations. If so how do you treat the double positives? B. 1st and 
> >2nd gate as above and then two separate dotpot CD4+ or CD8+ vs 
> >CD45RA and CD4+ or CD8+ vs CD45RO to get the % of all CD45RO+ on 
> >CD8+ and CD4+. C. 1st and 2nd gate as above and then using histogram 
> >instead of the last dotplots.
> >
> >Thank you for some suggestions and opinions, hoping to add some more 
> >spice to the discussion.
> >
> >Corrado
> >
> >Corrado M. Cilio, M.D., Ph.D.
> >Assistant Professor
> >Cellular Autoimmunity Unit
> >Dept. of Clinical Sciences/Paediatrics
> >Malmo University Hospital
> >Lund University
> >205 02 Malmo, Sweden
> >Tel: office +46-40332395; mobil: +46-70-4330338
> >"Judge a men by his questions rather than his answers" - Voltaire
> 
> 
>