Tregs from control and Esrrg-cKO splenocytes were isolated, either purified using Treg isolation kit II (Miltenyi Biotec) with the AutoMACS Pro (Miltenyi Biotec) or sorted as FOXP3-YFP+ with the S3e Cell Sorter (Bio-Rad). CD45.1+CD4+CD25– Teff cells from B6.SJL mice were labeled with CellTrace Violet (CTV) (Life Technologies, Thermo Fisher Scientific). Dendritic cells (DCs) were isolated from B6N mice by positive selection with anti-CD11c isolation kit (Miltenyi Biotec). CD45.2+CD4+CD25+ Tregs were incubated with Teff cells (6.6 × 104) at a 1:1 to 1:4 ratio in the presence of DCs (1 × 104) and soluble anti-CD3e antibody (1 μg/mL, BD Biosciences 145-2C11) for 3 days (74 (link)). The proliferation of CD45.1+ Teff cells was determined by the dilution of CTV, and the proliferation index was calculated with the FlowJo software.
For the experimental colitis model, B6.Rag–/– mice were injected i.v. with sorted CD4+FOXP3– Teff cells (0.4 × 106) either alone or with control or Esrrg-cKO Tregs (0.1 × 106). Mice were weighed and examined weekly and euthanized at week 13. Splenocytes were analyzed with flow cytometry, and colons were fixed and stained with H&E or anti-CD45 (1:25 dilution; BD Pharmingen 30F11). Infiltrating lymphocytes in colon were quantified with Imagescope.
For the experimental colitis model, B6.Rag–/– mice were injected i.v. with sorted CD4+FOXP3– Teff cells (0.4 × 106) either alone or with control or Esrrg-cKO Tregs (0.1 × 106). Mice were weighed and examined weekly and euthanized at week 13. Splenocytes were analyzed with flow cytometry, and colons were fixed and stained with H&E or anti-CD45 (1:25 dilution; BD Pharmingen 30F11). Infiltrating lymphocytes in colon were quantified with Imagescope.