Anti mil 4

Spleens were isolated and manipulated through a 40-µm filter, and erythrocytes were lysed with ACK lysis buffer. Different cell subsets were sorted by a fluorescence activated cell sorter (FACS) (Sony S800) according to the following cell markers: naïve CD4 (CD4⁺CD25⁻CD62L⁺CD44⁻); memory CD4 (CD4⁺CD25⁻CD62L⁻CD44⁺); Treg (CD4⁺CD25^hi); aTreg (CD4⁺CD25^hiCD44⁺CD62L⁻); rTreg (CD4⁺CD25^hiCD44⁻CD62L⁺); naïve CD8 (CD8⁺CD44⁻CD62L⁻); memory CD8 (CD8⁺CD44⁺); NK (Nkp46⁺); B cell (B220⁺); and macrophage (F4/80⁺CD11b⁺). Pan T cells were separated by negative selection using a Pan T Cell Isolation kit (Miltenyi Biotec, Germany). CD4⁺ and CD8⁺ T cells were isolated using anti-mouse CD4 and CD8 magnetic particles and separated by MACS.
For in vitro studies, cells were plated at a density of 5 × 10⁵ cells/well in 96-well plates precoated with anti-CD3 (Clone 500A2; BD Pharmingen) and anti-CD28 (clone 37.51; BD Pharmingen) (0.5 μg/ml) or stimulated with a cell stimulation cocktail (eBioscience). To polarize T cells toward a Th1 phenotype, 1 ng/ml mIL-12 (R&D) and 10 μg/ml anti–mIL-4 (BioLegend) were added to culture medium at the time of plating. For Th17 cells, 5 ng/ml mTGF-β, 20 ng/ml mIl-6, 2 ng/ml mIL-23 (Peprotech), 10 μg/ml anti-IFN-γ, and 10 μg/ml anti-IL-4 were added. For Treg induction, 5 ng/ml TGF-β, 100 IU rhIL-2, and 10 μg/ml anti-IFN-γ were added.