Lessons from humanized mice: Is Foxp3 a bona fide marker of human regulatory T cells?

Abstract

Abstract Regulatory T cells (Tregs) suppress conventional T cells (Tconv) and are critical for homeostasis of the immune system. Tregs are identified by the transcription factor Foxp3 and, to a large extent, by the transcription factor Helios (expressed by 80% of human Tregs). One confounding factor in the study of human Tregs is that Tconv cells upregulate Foxp3 expression upon activation in vitro. However, it remains unclear whether this is the case in vivo. To investigate differences in phenotype between Tregs and activated Tconv cells, peripheral blood mononuclear cells (PBMCs) from healthy human donors were activated in vitro and in vivo and analyzed for expression of activation markers and cytokines using flow cytometry. For in vitro activation, PBMCs were stimulated in xenogeneic reactions with mouse splenocytes or with anti-CD3. For in vivo activation, human PBMC-engrafted NOD-scid IL2Rgnull (NSG) mice were used. NSG mice develop xenogeneic graft-versus-host disease (GVHD) as human cells begin to attack mouse tissues. During in vitro activation, there was an increase in Foxp3 expression by IL-2 producing, Helios− Tconv cells. On the contrary, during the course of GVHD in vivo, activated Tconv cells, identified as CD25+ and Foxp3−, produced IL-2 but remained Foxp3− and Helios−. Foxp3+ Tregs did not produce IL-2 and remained predominantly Helios+. In conclusion, while expression of Foxp3 is not a reliable marker of activated human Tregs in vitro, it appears to be a specific marker of activated human Tregs in vivo as observed in this humanized mouse model. Using Foxp3 to distinguish human Tregs can improve diagnosis and treatment for autoimmune disorders and cancer.