Fig 1: GSDMD in peripheral myeloid cells is essential for the activation of CD4+ T Cells during EAE. (A) Schematic representation of the experiments in B, C, and Fig. 8 G. CD4+ T cells were obtained from spleens of WT or GSDMD−/− mice at day 12 after EAE induction, then transferred into Rag1−/− mice by i.v. injection or into WT mice by i.c.v. injection. DLN cells were obtained from WT or GSDMD−/− mice at day 12 after EAE induction, then cultured in vitro with MOG35-55 and IL-23 under Th17 cell–polarizing conditions before transfer into sublethal irradiated WTs or GSDMDfl/fl and GSDMDfl/flLysM-Cre mice, respectively. (B and C) Mean clinical scores after CD4 T cell and Th17 cell transfer by i.v. (B) and CD4 T cell transfer by i.c.v. (C; n = 5 mice per group). (D) Mean clinical scores after EAE induction in GSDMDfl/flLysM-Cre and littermate control GSDMDfl/fl mice (n = 5 mice per group); immunoblot analysis of GSDMD expression in BMDMs from indicated mice. (E) Summary graph of CNS-infiltrating immune cells at day 18 after immunization in GSDMDfl/flLysM-Cre and littermate GSDMDfl/fl mice (n = 5 mice per group). (F) Flow-cytometric analysis of KI67+ cells among CD4+ T cells in the CNS and spleens (SP) of GSDMDfl/flLysM-Cre and littermate GSDMDfl/fl mice at day 18 after immunization (n = 5 mice per group). (G) Immunofluorescent labeling of CD3 (red) and KI67 (green) demonstrate the proliferation of T cells in the spinal cord of indicated mice at day 18 after immunization. Data are presented as a representative image (left) and quantified percentage (right). Scale bar, 50 µm. (H) Quantified absolute cell numbers of CD4+ T cell in spleen and DLNs from GSDMDfl/flLysM-Cre and littermate control GSDMDfl/fl mice at day 18 after EAE induction (n = 4 mice per group). Data are pooled from three independent experiments (A–G) or from two independent experiments (H). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars show means ± SEM. Unpaired t test for B–D and F–H, and multiple unpaired t test for E.