title: Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level.
abstract: The transcription factor FOXP3 plays a key role in CD4(+)CD25(+) regulatory T cell function and represents a specific marker for these cells. Despite its strong association with regulatory T cell function, in humans little is known about the frequency of CD4(+)CD25(+) cells that express FOXP3 protein nor the distribution of these cells in vivo. Here we report the characterization of seven anti-FOXP3 monoclonal antibodies enabling the detection of endogenous human FOXP3 protein by flow cytometry and immunohistochemistry. Flow-cytometric analysis showed that FOXP3 was expressed by the majority of CD4(+)CD25(high) T cells in peripheral blood. By contrast, less than half of the CD4(+)CD25(int) population were FOXP3(+), providing an explanation for observations in human T cells that regulatory activity is enriched within the CD4(+)CD25(high) pool. Although FOXP3 expression was primarily restricted to CD4(+)CD25(+) cells, it was induced following activation of both CD4(+) and CD8(+) T cell clones. These findings indicate that the frequency of FOXP3(+) cells correlates with the level of expression of CD25 in naturally arising regulatory T cells and that FOXP3 protein is expressed by some activated CD4(+) and CD8(+) T cell clones. These reagents represent valuable research tools to further investigate FOXP3 function and are applicable for routine clinical use.
authors.affiliation: Monoclonal Antibodies Unit, Biotechnology Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Roncador
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Brown
authors.forename: Lorena
authors.initials: L
authors.lastname: Maestre
authors.forename: Sophie
authors.initials: S
authors.lastname: Hue
authors.forename: Jorge L
authors.initials: JL
authors.lastname: Martínez-Torrecuadrada
authors.forename: Khoon-Lin
authors.initials: KL
authors.lastname: Ling
authors.forename: Sarah
authors.initials: S
authors.lastname: Pratap
authors.forename: Christy
authors.initials: C
authors.lastname: Toms
authors.forename: Bridget C
authors.initials: BC
authors.lastname: Fox
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Cerundolo
authors.forename: Fiona
authors.initials: F
authors.lastname: Powrie
authors.forename: Alison H
authors.initials: AH
authors.lastname: Banham
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D003429:Cross Reactions; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000276:immunology; D051858:Forkhead Transcription Factors; D006801:Humans; D007108:Immune Tolerance; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D015375:Receptors, Interleukin-2 / Q000032:analysis*; D050378:T-Lymphocytes, Regulatory / Q000737:chemistry* / Q000502:physiology
chemical_list: D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; C498833:Foxp3 protein, mouse; D015375:Receptors, Interleukin-2