Fig. 4

ACT001 maintains microglial homeostasis and dampens their interactions with CD4⁺ T cells in vivo. (A-B) Flow cytometry analysis of active and homeostatic microglia in the SCs was performed at the peak of EAE, comparing ACT001-treated mice to controls (n = 3 per group). (A) Representative flow cytometry results of CD11b⁺CD45^Hi active microglia and CD11b⁺CD45^dim homeostatic microglia gated on Tmem119⁺. (B) Statistical analysis of the frequencies and absolute numbers of the microglia in (A). (C-D) Flow cytometry analysis of IA/IE (MHC-II) expression on microglia. (C) Representative flow cytometry results of MHC-II expression gated on Tmem119⁺ microglia. (D) Statistical analysis of the frequencies, absolute numbers, and mean fluorescence intensity (MFI) of MHC-II expression on microglia in (C). (E-F) Flow cytometry analysis of the interactions between Tmem119⁺ microglia and CD4⁺ T cells in single-cell (E) or doublet states (F), as well as the effect of ACT001 on this interaction. The Tmem119⁺CD4⁺ gate indicates the interacting microglia and CD4⁺ T cells. (G) Flow cytometry analysis of MHC-II expression by microglia in the doublets. The colors of the subtitles of (E-G) correspond to the colors in the gating strategy shown in Fig. S4C. (H) The frequencies of CD45^Hi microglia, MHC-II⁺ microglia, and CD4⁺ T cell-interacting microglia in single-cell or doublet states were statistically analyzed, along with the effects of ACT001. (I) Comparisons of the expression of MHC-II in Tmem119⁺CD4⁺ microglia and Tmem119⁺CD4⁻ microglia, as well as the effects of ACT001. Statistics were calculated using the unpaired sample t-test (B, D) and two-way ANOVA with Tukey’s correction (H). Error bars denote the mean ± SEM, ns = not significant, *p < 0.05, **p < 0.01 and ***p < 0.001