Figure 3. In vitro NOTCH2 stimulation... | Rockefeller University Press

Figure 3.

$Figure 3. In vitro NOTCH2 stimulation induces MZP cells toward an MZB-like profile. (A) Ex vivo naive, MZB, and MZP (MTG−MEM55+) cells were sorted from splenic samples of 3 children as described (see Materials and methods). In parallel, after a 2-d culture on OP9-hDLL1 cells, MZP cells having acquired a CD27 expression (p27 cells) were sorted as well, and their respective gene expression profiles were determined. The array dendrogram and heat-map expression profiles of naive, MZB, MZP, and p27, obtained by hierarchical clustering using the 1,855 probes that differentiated naive from MZB cells (P < 0.05, twofold higher expression), are shown. Each column represents microarray data from a sample of the indicated cell subtype and each row represents the expression of a single gene. Red squares indicate increased expression and green squares indicate decreased expression relative to the median expression of the gene according to the color bar shown (log2 scale). (B) Among the 1,855 probes discriminating MZB from naive B cells, those corresponding to cell surface proteins using the GSEA tool were determined, and the heat map expression profile of these surface markers are represented for each splenic sample. (C) Relative expression levels of SOX7, TOX, COCH, and HOPX (see Fig. 4) were determined by qPCR for the very fractions of naive, MZB, MZP, and p27 cells that have been used for the microarray-based gene expression analysis and for IgG+CD27+ cells from the same donors. Expression values normalized by B2M expression were calculated by the comparative method, normalizing to 1 the expression of the different genes in the MZB fraction. Mean values ± SEM of three independent experiments done in triplicates are shown. No differences were observed between p27 fractions and MZB cells. *, P < 0.05; n.s., nonsignificant; paired, two-tailed Student’s t test. (D) After gating on CD20+ cells, CD27-positive cells (p27) obtained after a 3-d culture of MZP on OP9-hDLL1 (red line) and ex vivo MZP (gray) and MZB (green) cells were analyzed for expression of several surface markers by flow cytometry, with GFP+ OP9 cells excluded from the analysis gate. Data are representative of 3 different samples.$

In vitro NOTCH2 stimulation induces MZP cells toward an MZB-like profile. (A) Ex vivo naive, MZB, and MZP (MTG⁻MEM55⁺) cells were sorted from splenic samples of 3 children as described (see Materials and methods). In parallel, after a 2-d culture on OP9-hDLL1 cells, MZP cells having acquired a CD27 expression (p27 cells) were sorted as well, and their respective gene expression profiles were determined. The array dendrogram and heat-map expression profiles of naive, MZB, MZP, and p27, obtained by hierarchical clustering using the 1,855 probes that differentiated naive from MZB cells (P < 0.05, twofold higher expression), are shown. Each column represents microarray data from a sample of the indicated cell subtype and each row represents the expression of a single gene. Red squares indicate increased expression and green squares indicate decreased expression relative to the median expression of the gene according to the color bar shown (log₂ scale). (B) Among the 1,855 probes discriminating MZB from naive B cells, those corresponding to cell surface proteins using the GSEA tool were determined, and the heat map expression profile of these surface markers are represented for each splenic sample. (C) Relative expression levels of SOX7, TOX, COCH, and HOPX (see Fig. 4) were determined by qPCR for the very fractions of naive, MZB, MZP, and p27 cells that have been used for the microarray-based gene expression analysis and for IgG⁺CD27⁺ cells from the same donors. Expression values normalized by B2M expression were calculated by the comparative method, normalizing to 1 the expression of the different genes in the MZB fraction. Mean values ± SEM of three independent experiments done in triplicates are shown. No differences were observed between p27 fractions and MZB cells. *, P < 0.05; n.s., nonsignificant; paired, two-tailed Student’s t test. (D) After gating on CD20⁺ cells, CD27-positive cells (p27) obtained after a 3-d culture of MZP on OP9-hDLL1 (red line) and ex vivo MZP (gray) and MZB (green) cells were analyzed for expression of several surface markers by flow cytometry, with GFP⁺ OP9 cells excluded from the analysis gate. Data are representative of 3 different samples.

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