Extended Data Figure 5. Aim2^fl/fl FGC R26T mice show normal T cell homeostasis, normal Treg proliferation and survival at steady state and during EAE.

a, Genotyping of FACS-sorted Treg (CD4⁺CD25⁺GFP⁺) and CD4 Tconv (CD4⁺CD25⁻) cells showing efficient deletion of Aim2 genomic DNA in Tregs. The image is representative of three independent experiments. b, Immunoblot analysis of AIM2 protein in Tregs from Aim2^+/+ FGC R26T and Aim2^fl/fl FGC R26T mice. The image is representative of three independent experiments. c, Images of Aim2^+/+ FGC R26T and Aim2^fl/fl FGC R26T mice and corresponding lymphoid organs of spleen and lymph nodes. d, Flow-cytometry of CD4⁺Foxp3⁺CD25⁺Tregs in the thymus, spleen and peripheral lymph node (PLN) of Aim2^+/+ FGC R26T and Aim2^fl/fl FGC R26T mice Representative results (upper) and statistical analysis (lower) of four experiments are shown. e, Flow-cytometry of CD4⁺ or CD8⁺ T cells in the spleen and PLN of Aim2^+/+ FGC R26T and Aim2^fl/fl FGC R26T mice. Representative results (left) and statistical analysis (right) of four experiments are shown. f-g, Flow-cytometry of naïve, effector/memory CD4⁺ (f) and CD8⁺ T cells (g) in the spleen and PLN of Aim2^+/+ FGC R26T and Aim2^fl/fl FGC R26T mice by assessing CD44 and CD62L expression. Representative results (left for f, upper for g) and statistical analysis (right for f, lower for g) of four experiments are shown. h-i, Flow-cytometry of IFNγ-, IL-4- or IL-17A-producing CD4⁺ cells (h) and IFNγ-producing CD8⁺ T cells (i) in Aim2^+/+ FGC R26T and Aim2^fl/fl FGC R26T mice. Representative results (upper) and statistical analysis (lower) of four experiments are shown. j, Statistical summary of IFNγ-, IL-17A-producing CD4⁺Tomato⁻ Tconv cells in the peripheral lymph node (PLN; left) and spleen (right) of Aim2^+/+ FGC R26T and Aim2^fl/fl FGC R26T mice at day 28 of an EAE course. Composite data summarized from three biological replicates. k, Statistical summary of IFNγ-, IL-17A-producing CD4⁺Tomato⁺ Tregs in the PLN (left) and spleen (right) of Aim2^+/+ FGC R26T and Aim2^fl/fl FGC R26T mice at day 28 of EAE course. Composite data summarized from three biological replicates. l, Flow-cytometry of Ki67 to analyze proliferation of Aim2^+/+ FGC and Aim2^fl/fl FGC Tregs in the PLN, spleen, and spinal cord (SC) during EAE. Left, representative sample; right, composite data summarized from three biological replicates. m, Apoptosis of Aim2^+/+ FGC and Aim2^fl/fl FGC Tregs in SC during EAE was analyzed by flow-cytometry using Annexin-V and 7AAD staining. Left, representative sample; right, composite data summarized from three biological replicates. n, Flow-cytometry of Ki67 to analyze proliferation of Aim2^+/+ FGC and Aim2^fl/fl FGC Tregs in the PLN and spleen at steady state. Left, representative sample; right, composite data of five mice of two independent experiments. o, Apoptosis of Aim2^+/+ FGC and Aim2^fl/fl FGC Tregs in the PLN and spleen at steady state was analyzed by flow-cytometry using Annexin-V and 7AAD staining. Left, representative sample; right, composite data of five mice of two independent experiments. Data are means ± SEM, P values: ns, not significant, analyzed by two-sided t test unless specified.