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

a, Genotyping of FACS-sorted T_reg (CD4⁺CD25⁺GFP⁺) and CD4⁺ T_conv (CD4⁺CD25⁻) cells showing efficient deletion of Aim2 genomic DNA in T_reg cells. The image is representative of three independent experiments. b, Immunoblot analysis of AIM2 protein in T_reg cells 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⁺T_reg cells in the thymus, spleen and PLN of Aim2^+/+ FGC R26T and Aim2^fl/fl FGC R26T mice Representative results (top) and statistical analysis (bottom) 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 naive, effector/memory CD4⁺ (f) and CD8⁺ (g) T cells 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, top for g) and statistical analysis (right for f, bottom 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 (top) and statistical analysis (bottom) of four experiments are shown. j, Statistical summary of IFNγ-, IL-17A-producing CD4⁺Tomato⁻ T_conv cells in the 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⁺ T_reg cells 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 analyse proliferation of Aim2^+/+ FGC and Aim2^fl/fl FGC T_reg cells in the PLN, spleen and spinal cord during EAE. Left, representative sample; right, composite data summarized from three biological replicates. m, Apoptosis of Aim2^+/+ FGC and Aim2^fl/fl FGC T_reg cells in the spinal cord during EAE was analysed 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 analyse proliferation of Aim2^+/+ FGC and Aim2^fl/fl FGC T_reg cells 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 T_reg cells in the PLN and spleen at steady state was analysed by flow cytometry using annexin V and 7AAD staining. Left, representative sample; right, composite data of five mice of two independent experiments. Data are mean ± s.e.m. P values determined by two-sided t-test unless specified.

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