FIGURE 5.

Treg-mediated Th17 differentiation requires activation of latent TGF-β1 via integrin αvβ8. (A) Naive CD45.1⁺ OT-II cells from Rag1^−/− mice were activated by soluble anti-CD3 in the presence of IL-6, splenic DCs, and preactivated CD4⁺CD25⁺ T cells from Itgb8 conditional knockout mice (CD4-CRE) or CRE-littermates. Cells were reactivated with Cell Stimulation Cocktail and Protein Transport Inhibitors and then stained for CD4, CD45.1, CD45.2, and IL-17A. The percentage of IL-17A⁺ cells derived from the CD4⁺CD45.1⁺CD45.2⁻cells in the culture are shown in each panel. (B) Same as in (A), except in the presence of IL-2 (100U/ml). Culture was stained for CD4, CD45.1, CD45.2, and intracellularly for Foxp3. (C and D) Same culture set up as in (A), except using preactivated CD4⁺CD25⁺ T cells from Itgb8 or Lrrc32 conditional knockout mice or CRE- littermates. In the right panel, half of the CD4⁺CD25⁺ T cells were from Itgb8 cKO mice and half were from Lrrc32 cKO mice. (D) Bar graph indicates the average (± SD) of duplicates within the experiment from (C). (E) Same as in (A), except using sorted CD4+Foxp3+ cells from Foxp3-GFP reporter mice and plate-bound anti-CD3 to activate the cells in the culture. Cells were cultured in the presence of a vehicle control (DMSO), the MMP-inhibitor GM6001, or the GM6001 control and measured for IL-17A as in (A).