Cd4 t cells

Joint draining LN cells from 6 week old KRN.g7, IDO1 ko KRN.g7, IDO2 ko KRN.g7, and dko KRN.g7 mice were harvested and stained for CD4⁺ T cells (BioLegend) and the following markers to distinguish T_H subsets: bcl6 (BD Bioscience), foxP3 (Biolegend), gata3, rorγt, T-bet (all from eBioscience) as described (1 (link)). The samples were acquired on a BD FACSCanto II flow cytometer using FACSDiva Software and analyzed using FlowJo Software.

Purified CD4⁺ T cells (> 95% purity) (Biolegend) were labeled with 1 µM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen) and rested or activated in RPMI 1640 medium (10% FBS) with human T-activator CD3/CD28 antibody (both 2 µg/ml, eBioscience) for 72 h. FACS-sorted NK cell subsets (CD56^dimCD57⁺ NK cells and CD56^dimCD57⁻ NK cells) were cocultured with these CD4⁺ T cells at a ratio of 1:1 for 15 h in RPMI 1640 medium (10% FBS) at 37℃ in a 5% CO₂ atmosphere. FACS analysis was performed after 15 h of coculture. Cytotoxicity was assessed by Fixable Viability Dyes (FVD) eFluor™ 780 (eBioscience). The percentage of FVD 780⁺ cells, gated on CFSE⁺ cells, was measured as the indicator of NK cell-mediated cytoxicity.