Optimal isolation of functional Foxp3+ induced regulatory T cells using DEREG mice.

2.50
Hdl Handle:
http://hdl.handle.net/10033/312331
Title:
Optimal isolation of functional Foxp3+ induced regulatory T cells using DEREG mice.
Authors:
Baru, Abdul Mannan; Untucht, Christopher; Ganesh, Venkateswaran; Hesse, Christina; Mayer, Christian T; Sparwasser, Tim ( 0000-0001-5645-902X )
Abstract:
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.
Citation:
Optimal isolation of functional Foxp3+ induced regulatory T cells using DEREG mice. 2012, 7 (9):e44760 PLoS ONE
Journal:
PloS one
Issue Date:
2012
URI:
http://hdl.handle.net/10033/312331
DOI:
10.1371/journal.pone.0044760
PubMed ID:
22957107
Type:
Article
Language:
en
ISSN:
1932-6203
Appears in Collections:
publications of the TwinCore unit Infection immunology

Full metadata record

DC FieldValue Language
dc.contributor.authorBaru, Abdul Mannanen
dc.contributor.authorUntucht, Christopheren
dc.contributor.authorGanesh, Venkateswaranen
dc.contributor.authorHesse, Christinaen
dc.contributor.authorMayer, Christian Ten
dc.contributor.authorSparwasser, Timen
dc.date.accessioned2014-02-06T14:10:18Zen
dc.date.available2014-02-06T14:10:18Zen
dc.date.issued2012en
dc.identifier.citationOptimal isolation of functional Foxp3+ induced regulatory T cells using DEREG mice. 2012, 7 (9):e44760 PLoS ONEen
dc.identifier.issn1932-6203en
dc.identifier.pmid22957107en
dc.identifier.doi10.1371/journal.pone.0044760en
dc.identifier.urihttp://hdl.handle.net/10033/312331en
dc.description.abstractFoxp3 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.en
dc.language.isoenen
dc.rightsArchived with thanks to PloS oneen
dc.subject.meshAnimalsen
dc.subject.meshCD4-Positive T-Lymphocytesen
dc.subject.meshCell Proliferationen
dc.subject.meshCell Separationen
dc.subject.meshChromosomes, Artificial, Bacterialen
dc.subject.meshDendritic Cellsen
dc.subject.meshFemaleen
dc.subject.meshFlow Cytometryen
dc.subject.meshForkhead Transcription Factorsen
dc.subject.meshGene Expression Regulationen
dc.subject.meshGenes, Reporteren
dc.subject.meshGenotypeen
dc.subject.meshGreen Fluorescent Proteinsen
dc.subject.meshInflammationen
dc.subject.meshInterleukin-2 Receptor alpha Subuniten
dc.subject.meshKineticsen
dc.subject.meshMaleen
dc.subject.meshMiceen
dc.subject.meshPromoter Regions, Geneticen
dc.subject.meshT-Lymphocytes, Regulatoryen
dc.titleOptimal isolation of functional Foxp3+ induced regulatory T cells using DEREG mice.en
dc.typeArticleen
dc.identifier.journalPloS oneen

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