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Association of two clones allows for optimal detection of human FOXP3.

ABSTRACT: FOXP3 is a key transcription factor expressed by regulatory T cells (Treg cells). However, differences in staining and analysis protocols have led to conflicting results. Moreover, the transient upregulation of FOXP3 that follows activation in non-Treg cells renders the interpretation of FOXP3 data more difficult in humans than in mice. Human peripheral blood mononuclear cells (PBMCs), isolated CD25(-) or CD25(+)CD4(+) T cells were stained with three different anti-FOXP3 clones (PCH101, 206D, and 259D) alone or in combination, and using different permeabilization methods. FOXP3 expression was evaluated following T cell activation by several pathways. Gating based on a population that did not express FOXP3 (such as CD3(-)CD4(-) T cells) allowed for the optimal characterization of Treg cells. The 206D clone detected a lower percentage of cells than PCH101 or 259D. In contrast, 259D stained a population of activated T cells that PCH101 did not. Staining with two clones together consistently increased the proportion of FOXP3(+) cells. However, it is likely that only the double positive cells are Treg cells, as they expressed the highest CD25 and lowest CD127 levels. Our results emphasize that the choice of staining protocol leads to very different results concerning the frequency of Treg cells in humans. A more consistent identification of these cells will improve the knowledge of their biology, particularly during disease processes.

SUBMITTER: Presicce P

PROVIDER: S-EPMC2903740 | biostudies-other | 2010 Jun

REPOSITORIES: biostudies-other

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Association of two clones allows for optimal detection of human FOXP3.

Presicce Pietro P Moreno-Fernandez Maria E ME Lages Celine S CS Orsborn Kris I KI Chougnet Claire A CA

Cytometry. Part A : the journal of the International Society for Analytical Cytology 20100601 6

FOXP3 is a key transcription factor expressed by regulatory T cells (Treg cells). However, differences in staining and analysis protocols have led to conflicting results. Moreover, the transient upregulation of FOXP3 that follows activation in non-Treg cells renders the interpretation of FOXP3 data more difficult in humans than in mice. Human peripheral blood mononuclear cells (PBMCs), isolated CD25(-) or CD25(+)CD4(+) T cells were stained with three different anti-FOXP3 clones (PCH101, 206D, an ...[more]

PMID: 20162533

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Project description:Foxp3 reporter mice including DEREG (DEpletion of REGulatory T cells) mice have greatly helped in exploring the biology of Foxp3(+) Tregs. DEREG mice express a DTR-eGFP fusion protein under the control of a bacterial artificial chromosome (BAC)-encoded Foxp3 promoter, allowing the viable isolation and inducible depletion of Foxp3(+) Tregs. Adaptive Tregs differentiated in vitro to express Foxp3 (iTregs) are gaining high interest as potential therapeutics for inflammatory conditions such as autoimmunity, allergy and transplant rejection. However, selective isolation of Foxp3(+) iTregs with a stable phenotype still remains to be a problem, especially in the human setting. While screening for culture conditions to generate stable CD4(+)Foxp3(+) iTregs from DEREG mice, with maximum suppressive activity, we observed an unexpected dichotomy of eGFP and Foxp3 expression which is not seen in ex vivo isolated cells from DEREG mice. Further characterization of eGFP(+)Foxp3(-) cells revealed relatively lower CD25 expression and a lack of suppressive activity in vitro. Similarly, eGFP(-) cells isolated from the same cultures were not suppressive despite of a broad CD25 expression reflecting mere T cell activation. In contrast, eGFP(+)Foxp3(+) iTregs exhibited potent suppressive activity comparable to that of natural eGFP(+)Foxp3(+) Tregs, emphasizing the importance of isolating Foxp3 expressing iTregs. Interestingly, the use of plate-bound anti-CD3 and anti-CD28 or Flt3L-driven BMDC resulted in considerable resolution of the observed dichotomy. In summary, we defined culture conditions for efficient generation of eGFP(+)Foxp3(+) iTregs by use of DEREG mice. Isolation of functional Foxp3(+) iTregs using DEREG mice can also be achieved under sub-optimal conditions based on the magnitude of surface CD25 expression, in synergy with transgene encoded eGFP. Besides, the reported phenomenon may be of general interest for exploring Foxp3 gene regulation, given that Foxp3 and eGFP expression are driven from distinct Foxp3 loci and because this dichotomy preferentially occurs only under defined in vitro conditions.

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Association of two clones allows for optimal detection of human FOXP3.

Publications

Association of two clones allows for optimal detection of human FOXP3.

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