Figure 2: Distinct spatial organization of pediatric Tregs in the extrafollicular space.
Immunofluorescence imaging of Tregs in adult (n=3 subjects, 12 images; 2–7 images per donor) and pediatric (aged < 3 years; n =3 subjects, 14 images; 3–8 images per donor) tonsil tissue. A) Representative pediatric (left) and adult (right) tonsil stained with antibodies for DAPI, CD4, CD8, FoxP3, CD19, and CD163. Sections shown at 40x magnification and orange rectangle box shown as outset at 160x magnification. Green arrows and circles denote Tregs (CD4+/FoxP3+). B) Analysis of density of Tregs within follicle and extrafollicular regions (see methods). Bars represent mean with standard deviation. Statistical testing with one-way ANOVA. C) Analysis of spatial interactions of Treg and all other immune cells (B cell, Macrophage, CD4 conventional (Tconv), Treg, CD8). Box represents interquartile range with bars representing mean and range. Statistical testing with unpaired t-test. D) Analysis of mean Treg to individual immune cells in extrafollicular regions based on randomly expected vs observed interactions (see methods). Box represent interquartile range with bars representing mean and range. Statistical testing with unpaired or Mann-Whitney t-test. B-D) Individual subjects are denoted by distinct shapes.
**= p <0.01, *** = p<0.001.