Fig. 6

Proliferative response of tonsillar B-cells from donors at different ages. (A) Freshly isolated TMC were cultured on CpG + CD40L + IL2 + IL4, for the time points indicated on the top of each panel. Cells were stained for surface CD20, CD4, CD8, CD73, CD39 and intra-nuclear Ki-67. Samples were subsequently analyzed by FACS. Gating strategy is illustrated. Singlets were gated by plotting FSC-H vs. FSC-A for each sample (upper panels). Within the singlets population, apoptotic and viable cells were distinguished by differences in forward and side scatter. Such scatter-based assay has good correspondence with results obtained by fluorescein isothiocyanate (FITC)- annexin staining for distinguishing the viability of untransformed human B cells [31, 32]. Within the viable gate, B cells were identified through the CD20 vs. CD4 dot plots. Dashed lines show the decline of expression of CD20 post stimulation. Proliferating B cells were recorded as the CD20⁺CD4⁻Ki-67⁺ cell population of the bottom dot plot panels and percentages designate the respective frequencies at each time point. Data from one donor representative of the 8 patients whose TMC were cultured. Gates were manually adjusted due to the changes experienced by the cells in culture upon each treatment. (B) Histograms presenting the mean percentage ± SD of the CD20⁺CD4⁻Ki-67⁺ population frequencies determined as in A), from 8 individuals, distributed according their age (4 children and 4 teenagers and adults). p value was calculated through unpaired t test, ns = con significant differences