Representative whole mount maximum projection confocal microscopy image of the (A) lumbar spinal cord meninges and (B) DRG showing Tregs (green-red: yellow) and nerves (autofluorescence, red) in Foxp3eGFP-CreERT2;Rosa26tdTomato reporter mice. Inlet showing DRG magnification. Scale bar represents 100 μm in A) and 150 μm in B). Arrows indicate Tregs. (C) Total number of weight-adjusted tissue Tregs across organs, in both sexes combined. (D) Relative number of tissue Tregs from male (white) and female (black) mice per organ. 100% represents mean number of female Tregs per organ. Comparison is made between each individual organ. (E) Representative concatenated flow cytometry plots of tissue Treg after a single intrathecal (IT) injection of 20 ng of pegylated diphtheria toxin (pegDT). (F) Relative quantifications of tissue Treg depletion 2 days after a single IT pegDT injection across organs. 100% represents mean number of tissue Tregs in IT vehicle-injected mice per organ. (G and H) 50% paw withdrawal thresholds measured using von Frey filaments before (day 0) and after a single dose of 20 ng of IT pegDT or vehicle in female (G) or male (H) Foxp3-DTR mice. (I) Summary of significant behavioral differences comparing IT pegDT- and control-injected female and male mice. Total number of mice for G-I is presented in Figure S3. ScMg= Spinal cord meninges, BrMg= Brain meninges, LN= Lymph nodes, ns = not significant, *p<0.05, **p<0.01, ***p<0.001. Related to
Figure S1-S3.