I am planning to characterise Treg using a large panel of markers. first I would like to subtype Treg either naïve, memory or activated, and that is just one basic question/ first step as I ll add many other markers for other purposes. so I would like to use one marker to answer this question if possible. Is it possible to subtype them using either CD45RO or CDRA or I need to use both for that and why?
Tregs tend to mirror the distribution of naive and memory T cell subsets observed for conventional T cells. Therefore, additional markers would help to delineate functional subsets of Tregs. While they are rare, a TEMRA-like Treg population does exist (CD45RA+CCR7-), and is not truly naive.
Many thanks Todd for ur answer.
For this reason, I am planning to use more than one activation marker such as CD39, CD38, CCR7, CD27 and HLADR in addition to the basic markers CD4, CD25, FOXP3, CD127low. I understand that Treg characterization is still complicated topic, but if u please have experience can u suggest if this is right way to do it?
You right. The characterization of T-reg is a very complex issue. Nevertheless, CD45RA+FOXP3+ T reg cells are a subset of naive thymus derived T-reg that upon activation lose CD45RA and acquire CD45RO. Conversely FOXP3+CD45RO+ can be either activated thymus derived T-reg or conventional activated T-cells with no regulatory activity. Regarding your question, the use of both can add some more information. Hope to be of any help
Hi, you might also add CCR4 to your panel of activation markers. Sakaguchi's team (Sugiyama et al., PNAS 2013, PMID: 24127572) claims that CCR4 is expressed by a subset of "the most effector/supressive" CD45RA-FOXP3hi Tregs but not by CD45RA+FoxP3low "naive" Tregs. Hope it helps.
Hi Fatma,
As suggested above identification of human Tregs remains difficult but the CD45RA and Foxp3 lo/hi method seems as good as anything else. Details in Miyara et al. Immunity. 2009 Jun 19;30(6):899-911.
I confess I'm more of a mouse Treg guy myself but I share an office with the human Treg arm of the Sakaguchi group. Sugiyama-san the first author of the CCR4 manuscript is of the opinion that CCR4 is a good treatment target but for identification CD45RA and Foxp3 are still the gold standard as the Foxp3lo non Tregs do express CCR4 but at a lower level than the Foxp3hi eTregs (see figure 1 of the PNAS article). Although more markers don't hurt if you have space.
Best regards, James
I believe you can find your answers from the publication below on human Treg. The interest lies in the functional characterization of subsets of Treg present in Human PBMCs.
FoxP3hiCD45- as well as FoxP3loCD45+ are
Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor.
PMID: 19464196
http://www.ncbi.nlm.nih.gov/pubmed/19464196
There id also this good review:
The Phenotypic Markers of CD4+CD25+ T Regulatory Lymphocytes
Jan Lastovicka
2nd Medical School and Faculty Hospital Motol, Czech republic
Volume 2013 (2013), Article ID 119348, Research in Immunology: An International Journal , 14 pages, DOI: 10.5171/2013.119348
Received date: 7 August 2013; Accepted date: 12 September 2013
Published date: 14 November 2013
Academic Editor: Jolanta Myśliwska
http://www.ibimapublishing.com/journals/IMMU/2013/119348/119348.pdf
Good luck!
I just want to add in, as long as you are using human adult PBMC subsets, using CD45RA/RO or CD45RA/CD27 works well. Keep in mind CCR7 status does work in place of CD27, but if you are using frozen cells, CCR7 is sheared by the freeze/thaw and is thus not a good marker. Also, if you are for example using human cord blood T cell subsets, you will absolutely have to use CD27 in addition to CD45RA/RO as CB T cells can be memory and still be CD45RA+ (TEMRA CD45+CD27-).
Hi Recently there was a publication that summarizes the different definitions of Treg in an immune monitoring context. Check out, Santegoets et al., Cancer Immunol Immunother (2015) 64:1271–1286. Based on this paper, I gate CD45RA- Ki67+ cells as activated Treg and CD45RA- Ki67- cells as memory Treg. Both these populations are first gated on CD25+FoxP3+ bulk Treg. Hope it helps!
Very useful insight reading all the comments. Like Fatma, I am working on human T regs and have been in a bit of a dilemma as to what markers to use to delineate the functional subset of Tregs. I was going to use CD45RA and Ki-67 but seems like CCR4 is a good one to add in to the mix too, given that it is a recommendation from the Sakaguchi group.
The other dilemma I have is whether to stick to CD4 CD25 FoxP3 or whether it is necessary to also use CD127. I note that Fatma hasn't mentioned Foxp3 in the panel. Any reasons why? I thought this was crucial in identifying the Treg population.
Hi Mildred, sorry I haven't share what markers I decided to use in my panel. I did use CD4 CD25 and FOXP3 to identify Treg.
Great, thanks Fatma. What markers did you finally use for identifying the functional subset?
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