Fig. 1.

Isolation and characterization of the Il2ra^mut/mut mouse model. a Lymph node cells isolated from WT, Ccr2^−/−2009 and Ccr2^−/−2012 (all on the B6 background) mice were stained with mAbs against cell-surface CD3, CD4, CD25 (clone PC61), and intracellular Foxp3. A representative dot plot is shown. Bar graph summarizes CD25 expression levels (MFI) across all mice analyzed. b FACS histograms of cell-surface CD25 levels (MFI) gated on blood Foxp3⁺ T_reg cells (CD3⁺CD4⁺) from Ccr2^−/−2009, Ccr2^−/−2012, Ccr2^+/−2009 (F1) mice. c Distribution of CD25 cell-surface expression levels (MFI) in T_reg cells from the blood of individual mice of indicated genotypes including Ccr2^+/−2009 × Ccr2^+/−2009 (F2) mice. d Ccr2 genotypes based on CD25 surface expression levels and experimental frequencies obtained in F2 offsprings. e DNA and corresponding amino-acid sequence alignments of the mutation found in exon 4 of Il2ra gene in Ccr2^−/−2009 vs Ccr2^−/−2012 mice after whole-exome sequencing. f Alignment of the CD25 amino-acid sequence surrounding tyrosine 129 across multiple species. g FACS histograms of cell-surface expression of CD25 in 293T cells retrovirally transduced with WT or mutagenized Y129H Il2ra cDNA. Bar graph shows CD25 expression (MFI) across two independent transduction experiments. h Dot plots of cell-surface or intracellular expression levels (MFI) of CD25 in WT and mutant T_reg cells. Bar graphs average data from one of 2–3 experiments with similar results with >2 mice per group. i Fluorescent microscopy staining of CD25 on anti-CD3 stimulated purified CD4⁺ T cells isolated from LNs. T cells were fixed and co-stained with anti-CD25 (green) and anti-calreticulin (red) prior to image acquisition. Scale bar is 5 μm. p-values are indicated when relevant with *p < 0.05; **p < 0.01; ***p < 0.001; NS not significant, using two-tailed unpaired Student’s t-test. Error bars are mean ± SEM in all figures