Cutting edge: the Th1 response inhibits the generation of peripheral regulatory T cells

Abstract

The possibility that effector T cells can be converted into forkhead box P3(+) regulatory T cells (Tregs) has potential therapeutic implications. To analyze the relationship between Th1 effectors and Tregs, we have used a model of systemic autoimmunity in which both effector and Tregs arise from a single population specific for a transgene-encoded systemic protein. In vitro, the presence of IFN-gamma inhibits Treg generation during activation. Using IFN-gamma reporter mice, we demonstrate that IFN-gamma-producing cells tend not to develop into Tregs, and Th1 priming of T cells prior to cell transfer limits the number of forkhead box P3(+) T cells generated in vivo. Moreover, transfer of IFN-gamma(-/-) or STAT1(-/-) T cells resulted in an increase in the number of Tregs. These data support a role for Th1 effector molecules and transcription factors in the control of peripheral Treg generation and demonstrates the limited plasticity of Th1 populations.

FIGURE 1

Relationship of IFN-γ–producing effector cells and Foxp3⁺ Tregs. A, Naive CD4⁺ T cells from DO11.10 Yeti mice were sorted and adoptively transferred into sOVARag^−/− recipients. Lymph nodes were harvested 10 d post transfer. Representative FACS plots of IFN-γ and Foxp3 gated on DO11.10 cells. B, Cumulative data from four experiments for the percentage of IFN-γ⁺ or Foxp3⁺ cells; note the difference in scales. C, Naive T cells from DO11.10 Foxp3-GFP reporter mice were sorted and adoptively transferred into sOVARag^−/− recipients. GFP⁺ Tregs cells were sorted 10 d post transfer (d10 Tregs) and compared with GFP⁺ Tregs from DO11.10 Foxp3-GFP reporter mice (natural). These cells were cultured with CD4⁺CD25⁻ (responder) cells from DO11.10/Rag^−/− mice and mitomycin-treated APCs for 72 h. Data are representative of two comparable experiments.

FIGURE 2

Reduced Treg development from IFN-γ–producing cells. Naive CD4⁺ T cells from DO11.10 Yeti mice were sorted and cultured under Th1 conditions. After 5 d, cells were sorted into YFP⁻ and YFP⁺ populations and transferred into sOVARag^−/− recipients; for comparison, freshly sorted naive cells were also transferred. Lymph nodes were harvested 10 d post transfer. A, Representative FACS plots show Foxp3 expression gated on DO11.10 cells. B, The percentage of Foxp3⁺ T cells for three experiments (note the difference in scales). C, The number of Foxp3⁺ T cells for three experiments. D, Naive CD4⁺ T cells from WT DO11.10 mice were sorted and cultured under Th1 conditions for three rounds of stimulation (3R Th1) and transferred into sOVARag^−/− recipients. Lymph nodes were harvested 10 d post transfer. Representative FACS plots are shown gated on DO11.10 cells. E, The number of Foxp3⁺ T cells for three experiments.

FIGURE 3

IFN-γ inhibits development of Foxp3⁺ cells. A, Naive T cells from WT and IFN-γ^−/− DO11.10 mice were cultured in activating conditions for 5 d and then transferred to sOVARag^−/− recipients; for comparison, freshly sorted naive cells were also transferred. Lymph nodes were harvested 10 d post transfer. A, Representative plots of Foxp3⁺ T cells gated on DO11.10 cells. B, The percentage of Foxp3⁺ T cells for three experiments. C, The number of Foxp3⁺ T cells for three experiments. D, Naive CD4⁺ T cells from WT and IFN-γ^−/− DO11.10 mice were cultured in activating conditions ± exogenous IFN-γ for Foxp3 expression on days 2, 4, and 6. Data are representative of two experiments.

FIGURE 4

STAT1 but not Tbet inhibits peripheral Treg generation. Naive CD4⁺ T cells from WT, Tbet^−/−, or STAT1^−/− DO11.10 mice were sorted, cultured under Th1 conditions, and transferred to sOVARag^−/− recipients. Lymph nodes were harvested 10 d post transfer. A, Representative plots of Foxp3 stains gated on DO11.10. B, The percentage of Foxp3⁺ T cells for three experiments. C, The number of Foxp3⁺ T cells from three experiments.