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. Author manuscript; available in PMC: 2026 Oct 2.
Published in final edited form as: Immunity. 2025 Oct 2;58(11):2753–2768.e6. doi: 10.1016/j.immuni.2025.09.007

Fig. 4. Notch3 destabilizes Treg cells to promote EAE.

Fig. 4.

A and B. Volcano plot (A) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (B) of gene transcripts of CNS Notch3-sufficient (Foxp3YFPCre, n= 4, pool of 16 mice) vs. Notch3-deficient (Foxp3YFPCreNotch3Δ/Δ, n= 4, pool of 16 mice) Treg cells. C, Flow cytometric analysis of Treg (YFP+RFP+), ex-Treg (YFP+RFP–) and Tconv cells (YFP–RFP–) in Foxp3YFPCreR26LSL-RFP and Foxp3YFPCreNotch3Δ/ΔR26LSL-RFP mice and frequencies of ex-Treg cells in control (PBS) vs. Foxp3YFPCreR26LSL-RFP and Foxp3YFPCreNotch3Δ/ΔR26LSL-RFP mice at day 15 after EAE induction. D, Frequencies of ex-Treg cells in different tissue in Foxp3YFPCreR26LSL-RFP mice at day 15 after EAE induction. E. Flow cytometric analysis and frequencies of Notch3+ Treg, ex-Treg and Tconv cells in Foxp3EGFPCreR26YFP and Foxp3YFPCreNotch3Δ/ΔR26LSL-RFP mice in control (PBS) vs. EAE mice. F. Flow cytometric analysis and frequencies of Yap1 positive Treg cells in Foxp3YFPCre vs. Foxp3EGFPCreNotch3Δ/Δ Treg cells in EAE. G and H. Time scores (G), cumulative and maximal scores (H) of EAE in Foxp3YFPCre, Foxp3YFPCreNotch3Δ/Δ or Foxp3YFPCreYap1Δ/ΔWwtr1Δ/Δ mice. I. Notch3 expression in Treg and Foxp3– cells in Foxp3YFPCre vs. Foxp3YFPCreYap1Δ/ΔWwtr1Δ/Δ mice in EAE. J, Frequencies of ex-Treg cells in Foxp3YFPCreR26LSL-RFP and Foxp3YFPCreYap1Δ/ΔWwtr1Δ/ΔR26LSL-RFP mice at day 15 after EAE induction. K and L. IFNγ+IL17+, IFNγ–IL-17+(K) and IL-22+IL-17+ (L) CD4+ T cells in the respective mouse strains at day 15 post-EAE. M. Flow cytometric analysis of Foxp3 expression and frequencies of Foxp3+ Treg cells in co-cultures of CNS Treg cells derived from MOG33-55-immunized Foxp3YFPCre, Foxp3YFPCreNotch3Δ/Δ and Foxp3YFPCreYap1Δ/ΔWwtr1Δ/Δ mice and co-cultured with microglial cells of MOG33-55-immunized Foxp3YFPCre mice. Panels C-I and K-M represent a pool of three experiments, panels J represent one experiment. Error bars are SEM. Each point denotes one mouse. Statistical analysis: student two-tailed t-test (F, I, J, K, L) one-way ANOVA with Dunnett’s post-hoc analysis (C, D, H, M) or two-way ANOVA with Sidak post-hoc analysis (E).