Fig. 4

Teenagers and adults’ tonsils comprise less proliferative cells than children. (A) Representative immunohistochemistry staining of Ki-67 on tonsils biopsies from donors of the indicated ages at two different magnifications (also indicated). Scale bar, 500 and 200 μm, respectively. (B) Fresh TMC were stained for surface CD4, CD20 and CD3, also for intra-nuclear Ki-67 and a fixable viability dye. Samples were subsequently analyzed by FACS. The gating strategy to identify the proliferating B and CD4⁺ T cell populations is partially illustrated. Singlets were gated by plotting FSC-H vs. FSC-A for each sample (not shown). Within the singlets population, dead cells were determined by the fixable viability dye (not shown). Within the viable gate, lymphoid gate was determined through SSC-A vs. FSC-A (not shown). Left panels: dot plots depicting the percentage of the tonsillar CD20⁺Ki-67⁺ subsets from single donors of the indicated ages. Right panels: dot plots depicting the percentage of the tonsillar CD3⁺CD4⁺Ki-67⁺ subsets from single of the indicated ages. Percentages designate frequencies of the populations indicated. (C) Histograms presenting the mean percentage ± SD of the cell population frequencies determined as in B) from 60 individuals distributed according their age. p value was calculated through unpaired t test, *p < 0.05; **p < 0.01 and **** p < 0.0001