Identification of candidate MZPs from children spleen. (A) Isolation scheme of candidate MZP subsets from MNCs from children spleens (4–8 yr). (B) MTG and MEM55 (an antibody against a glycosylated variant of CD45RB) flow cytometry profiles of IgG⁻IgE⁻IgA⁻CD27⁻CD20⁺ cells revealed four distinct fractions that were isolated after the indicated CD24 and CD38 sort gates. (C) The four fractions isolated in B were co-cultured either with OP9 + hDLL1 or OP9 cells alone. After 3 d, cells were recovered, stained for CD20, IgD, and CD27, and CD20⁺-gated cells were analyzed for CD27 up-regulation. For MTG⁻MEM55⁺ and MTG⁺MEM55⁺ cells, the effect of a blocking anti-NOTCH2 mAb added in the OP9-hDDL1 cultures was evaluated. Percentages of IgD⁺CD27⁺ among total B cells after differentiation are indicated on each dot plot. Mean B cell survival after 3 d of culture was 19 ± 3% for both MEM55⁺ subsets. Experimental repeats (3 or 5 depending on the assay) are depicted in Table S1. (D) For the two MEM55⁺ subsets, a fold induction corresponding to the ratio of IgD⁺ cells becoming CD27-positive in the OP9-hDDL1 (with or without anti-NOTCH2 mAb) versus OP9 condition were plotted with indicated means ± SD (complete data from 5 different donors are listed in Table S1). Statistical significance is indicated on graphs as determined by paired, two-tailed Student’s t test. **, P < 0.01; n.s., nonsignificant.