Fig. S1

Lymphocyte expansion and lymphocyte-dependent inflammation in foxp3a mutants. (A) Flow cytometry analyses of WKM from representative foxp3a mutant animals. (B) Differential counts of nonerythroid cells from WKM. Counts were obtained from at least 200 cells per WKM cytospin with May-Grünwald Giemsa staining. Two-tailed Student’s t test (foxp3a mutant vs. AB), *, P < 0.05; **, P < 0.001. (C) Representative images of hematoxylin and eosin–stained sagittal sections of spleens from wild-type AB and foxp3a(um252) mutants. Images showing largest extent of splenic size are shown. Spleens are shown bordered by dashed lines. Bars, 250 µm. (D) Representative images of dissected spleens from Tg(lck:EGFP) and Tg(lck:EGFP); foxp3a(um252) animals. Spleens are shown bordered by dotted lines. Bars, 250 µm. (E) Flow cytometry analysis of spleens from AB (n = 5), Tg(lck:EGFP) (n = 5) and Tg(lck:EGFP); foxp3a(um252) (n = 5) animals. Percentages of EGFP-positive cells in the lymphocyte gate are shown. Two-tailed Student’s t test, *, P < 0.05. (F) Representative images of hematoxylin and eosin–stained sagittal sections of urogenital pores from wild-type AB, foxp3a(um252), and foxp3a(um252); rag1(lf) animals. In total, 0/4 wild-type AB, 6/6 foxp3a(um252), and 0/3 foxp3a(um252); rag1(lf) animals displayed connective tissue inflammation. Insets, increased cellularity characterized by marked influx of lymphocyte-predominant inflammatory cells (arrowheads) in foxp3a(um252) mutants compared with the wild-type AB and foxp3a(um252); rag1(lf) double mutants. Bars: 100 µm; (inset) 10 µm. (G) Representative images of hematoxylin and eosin–stained sagittal sections of skin from wild-type AB, foxp3a(um252), and foxp3a(um252); rag1(lf) animals. In total, 0/4 wild-type AB, 5/6 foxp3a(um252), and 0/3 foxp3a(um252); rag1(lf) animals displayed skin inflammation. Bar, 25 µm. (H) qRT-PCR analysis of inflammation marker genes ifng1-2 and il1b. ΔCt values were calculated relative to a β-actin control. Two-tailed Student’s t test, *, P < 0.05; ns, not significant. Error bar indicates SEM; n = 3.