title: Phenotypic and functional characterization of a CD4(+) CD25(high) FOXP3(high) regulatory T-cell population in the dog.
abstract: Relatively little is known about regulatory T (Treg) cells and their functional responses in dogs. We have used the cross-reactive anti-mouse/rat Foxp3 antibody clone FJK-16s to identify a population of canine CD4(+) FOXP3(high) T cells in both the peripheral blood (PB) and popliteal lymph node (LN). FOXP3(+) cells in both PB and LN yielded positive staining with the newly developed anti-murine/human Helios antibody clone 22F6, consistent with the notion that they were naturally occurring Treg cells. Stimulation of mononuclear cells of LN origin with concanavalin A (Con A) in vitro yielded increased proportions and median fluorescence intensity of FOXP3 expression by both CD4(+) and CD8(+) T cells. Removal of the Con A and continued culture disclosed a CD4(+)  FOXP3(high) population, distinct from the CD4(+) FOXP3(intermediate) T cells; very few CD8(+) FOXP3(high) T cells were observed, though CD8(+) FOXP3(intermediate) cells were present in equal abundance to CD4(+) FOXP3(intermediate) cells. The CD4(+)  FOXP3(high) T cells were thought to represent activated Treg cells, in contrast to the FOXP3(intermediate) cells, which were thought to be a more heterogeneous population comprising predominantly activated conventional T cells. Co-staining with interferon-γ (IFN-γ) supported this notion, because the FOXP3(high) T cells were almost exclusively IFN-γ(-) , whereas the FOXP3(intermediate) cells expressed a more heterogeneous IFN-γ phenotype. Following activation of mononuclear cells with Con A and interleukin-2, the 5% of CD4(+) T cells showing the highest CD25 expression (CD4(+) CD25(high) ) were enriched in cells expressing FOXP3. These cells were anergic in vitro, in contrast to the 20% of CD4(+) T cells with the lowest CD25 expression (CD4(+) CD25(-) ), which proliferated readily. The CD4(+) CD25(high) FOXP3(high) T cells were able to suppress the proliferation of responder CD4(+) T cells in vitro, in contrast to the CD4(+) CD25(-) cells, which showed no regulatory properties.
authors.affiliation: Regulatory T Cell Laboratory, Department of Veterinary Clinical Sciences, The Royal Veterinary College, London, UK.
authors.forename: Dammy
authors.initials: D
authors.lastname: Pinheiro
authors.forename: Yogesh
authors.initials: Y
authors.lastname: Singh
authors.forename: Charlotte R
authors.initials: CR
authors.lastname: Grant
authors.forename: Richard C
authors.initials: RC
authors.lastname: Appleton
authors.forename: Flavio
authors.initials: F
authors.lastname: Sacchini
authors.forename: Kate R L
authors.initials: KR
authors.lastname: Walker
authors.forename: Alden H
authors.initials: AH
authors.lastname: Chadbourne
authors.forename: Charlotte A
authors.initials: CA
authors.lastname: Palmer
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Armitage-Chan
authors.forename: Ian
authors.initials: I
authors.lastname: Thompson
authors.forename: Lina
authors.initials: L
authors.lastname: Williamson
authors.forename: Fiona
authors.initials: F
authors.lastname: Cunningham
authors.forename: Oliver A
authors.initials: OA
authors.lastname: Garden
mesh_terms: D000818:Animals; D015704:CD4 Antigens / Q000276:immunology*; D002478:Cells, Cultured; D004285:Dogs; D051858:Forkhead Transcription Factors / Q000276:immunology*; D053645:Interleukin-2 Receptor alpha Subunit / Q000276:immunology*; D010641:Phenotype; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000276:immunology* / Q000378:metabolism*
chemical_list: D015704:CD4 Antigens; D051858:Forkhead Transcription Factors; D053645:Interleukin-2 Receptor alpha Subunit