Murine CD4+ T cells were negatively selected from the cells of the spleen and lymph nodes. For the preparation of effector T cells, regulatory T cells were depleted from the CD4+ T cells using biotin-conjugated anti-mouse CD25 antibody (clone PC61, BioLegend) by MACS magnetic-bead separation (Miltenyi Biotec). For purification of nTreg cells, nTreg cells were isolated from CD4+ T cells using biotin-conjugated anti-mouse Nrp-1 antibody (clone 3E12, BioLegend) by MACS magnetic-bead separation (Miltenyi Biotec). For purification of the in vitro differentiated iTreg cells, iTreg cells were isolated from the in vitro differentiated CD4 cells using biotin-conjugated anti-mouse CD25 antibody (clone PC61, BioLegend) by MACS magnetic-bead separation (Miltenyi Biotec). The purity of iTreg (CD4+Foxp3+) cells was 90% (Figure S1B). For determination of effector T cell proliferation, effector T cells were labeled with 1 μM CFSE (Thermo Fisher Scientific). 5105 CFSE-labelled effector T cells were co-cultured with 1.66105 Treg cells for 4 days under the stimulation of anti-CD3 antibody (1 μg/ml, BD Biosciences) coated on 96-well round-bottom plates.
Chi J.N., Yang J.Y., Hsueh C.H., Tsai C.Y., Chuang H.C, & Tan T.H. (2022). MAP4K3/GLK inhibits Treg differentiation by direct phosphorylating IKKβ and inducing IKKβ-mediated FoxO1 nuclear export and Foxp3 downregulation. Theranostics, 12(13), 5744-5760.
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