Induction of Regulatory T Cells by Kynurenine-Treated Dendritic Cells

LPS-unprimed and LPS-primed cDCs treated or not with L-kynurenine, as described above, were cultured with CD4⁺ T cells isolated from the spleens of Foxp3^YFP mice. Briefly, CD4⁺ T cells were isolated via cell separation by two steps. A first step was meant to enrich the CD3⁺ cell fraction, using biotin mouse monoclonal antibodies against B220 (a marker of pDCs and B cells) and CD11c (a marker of DCs). After this step, cells were incubated with MagniSort Streptavidin Negative Selection Beads (Thermo Fisher) followed by depletion of B220⁺CD11c⁺. B220^– CD11c^– collected cell fractions were incubated with magnetic anti-mouse CD4 beads (Miltenyi Biotec) to select CD4⁺ T cells. The purity of CD4⁺ T cells was verified by FACS analysis by cell staining with anti-B220, anti-CD3, anti-CD11c, anti-CD4, and anti-CD8 specific antibodies. CD4⁺ T cells (2 × 10⁵) were activated with 5 μg/ml anti-CD3 mAb (clone OKT3) and co-cultured for 4 days with unprimed and primed cDCs (5 × 10⁴) treated or not with L-kynurenine for 36 h (19 (link)). Treg cells were evaluated by FACS, analyzing the induction of Foxp3 expressing YFP.