Characterization of stable human CD4+ T cell clones that constitutively express high levels of CD25 and FOXP3.

In humans, FOXP3 expression is stable and constitutively high in the CD4+CD25+ T cells that display suppressive function in vitro, but it is also transiently upregulated in activated CD4+CD25- T cells. It was recently shown that CpGs located in FOXP3 intron 1 are unmethylated in both murine and human CD4+CD25+ regulatory T cells, in contrast to what is observed in activated conventional T cells. We selected FOXP3hi clones derived from cancerous or non-cancerous patients on the basis of the unmethylated status of the intronic region of gene FOXP3. These human FOXP3hi clones produce no IL-2, IFNg, TNFa, IL-4, IL-5 or IL-10. They suppress the proliferation of activated T cells in vitro, and at least part of this suppressive function can be attributed to the reduction of growth factor availability in the co-culture. We analyzed the gene expression profiles of activated FOXP3hi or conventional CD4+ clones by Affymetrix microarray. Approximately half of the genes specifically up-regulated in activated FOXP3hi T clones are also upregulated by TGFbeta in conventional CD4+ T cell clones. Moreover, half of the genes upregulated as a result of activation in conventional CD4+ but not in FOXP3hi clones, can be downregulated by TGFbeta. It appears therefore that a significant proportion of the gene expression profile of activated human FOXP3hi clones corresponds to a TGFbeta signature. In addition, a “suppressed” T clone co-cultured with a FOXP3hi clone also shows a TGFbeta signature. We propose that activated human FOXP3hi T cells suppress the proliferation of neighbouring T cells via a combination of growth factor consumption and bioactive TGFbeta production. Interestingly, TGFbeta also acts in an autocrine manner on FOXP3hi cells, potentially participating to their inability to produce growth factors