Figure 2. The MTG − MEM55 subset... | Rockefeller University Press

Figure 2.

$Figure 2. The MTG−MEM55+ subset represents an early MZP cell stage before Ig gene diversification. (A) Gene expression profiles of naive, MZB, MTG−MEM55+, and MTG+MEM55+ cells were determined using Affymetrix Human Genome U133 2.0 Plus microarrays (see Materials and methods). A 3D representation of a principal component analysis (PrC) performed for these four subsets, based on the expression of the 1,855 probes that were differentially expressed between naive and MZB cells, with a fold change ≥2 (P < 0.05), is shown, with each dot representing a single sample. (B) Mutation distribution within Ig sequences from splenic MZB, MTG+MEM55+, and MTG−MEM55+ subsets and mutation frequencies per total sequences. Mutations were analyzed within 284 pb of rearranged JH4-JH5 intronic sequences with data pooled from five children (detailed in Fig. S1). The pie charts depict relative proportions of sequences with a given mutation range (see the color legend). The total number of analyzed sequences is indicated in the center of each chart. Mutation frequencies of all sequences from each subset are represented, expressed as mutations per 100 bp (mean of 5 splenic samples ± SEM). **, P < 0.01; *, P < 0.05; paired, two-tailed Student’s t test. (C) Relative expression levels of SOX5 were determined by qPCR for naive, MZB, MTG+MEM55+, and MTG−MEM55+ cells. 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. **, P < 0.01; *, P < 0.05; paired, two-tailed Student’s t test. (D) Through data mining, a list of 1,678 and 1,453 genes that were, respectively, up or down-regulated after induction of an activated NOTCH2 receptor (NOTCH2-IC) in an EBV-transformed human B lymphoblastoid cell line (LCL) was established. Those genes were compared with the 215 and 154 genes that were, respectively, over- or under-expressed in MZP compared with naive cells (with a fold change ≥1.5, P < 0.05) to generate Venn diagrams. The numbers of genes that are common in both lists are indicated in bold at the intersection between two circles. Hypergeometric distribution was used to calculate the p-value according to GSEA. (E) After gating on dump−CD19+ cells, and based on their respective expression of MTG, MEM55, CD27, and IgD, naive (gray), MZB (red), MTG−MEM55+ (blue), and MTG+MEM55+ (black, dotted line) cells from children spleen samples were analyzed for expression of several surface markers by flow cytometry. Data are representative of 3 different samples.$

The MTG⁻MEM55⁺ subset represents an early MZP cell stage before Ig gene diversification. (A) Gene expression profiles of naive, MZB, MTG⁻MEM55⁺, and MTG⁺MEM55⁺ cells were determined using Affymetrix Human Genome U133 2.0 Plus microarrays (see Materials and methods). A 3D representation of a principal component analysis (PrC) performed for these four subsets, based on the expression of the 1,855 probes that were differentially expressed between naive and MZB cells, with a fold change ≥2 (P < 0.05), is shown, with each dot representing a single sample. (B) Mutation distribution within Ig sequences from splenic MZB, MTG⁺MEM55⁺, and MTG⁻MEM55⁺ subsets and mutation frequencies per total sequences. Mutations were analyzed within 284 pb of rearranged J_H4-J_H5 intronic sequences with data pooled from five children (detailed in Fig. S1). The pie charts depict relative proportions of sequences with a given mutation range (see the color legend). The total number of analyzed sequences is indicated in the center of each chart. Mutation frequencies of all sequences from each subset are represented, expressed as mutations per 100 bp (mean of 5 splenic samples ± SEM). **, P < 0.01; *, P < 0.05; paired, two-tailed Student’s t test. (C) Relative expression levels of SOX5 were determined by qPCR for naive, MZB, MTG⁺MEM55⁺, and MTG⁻MEM55⁺ cells. 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. **, P < 0.01; *, P < 0.05; paired, two-tailed Student’s t test. (D) Through data mining, a list of 1,678 and 1,453 genes that were, respectively, up or down-regulated after induction of an activated NOTCH2 receptor (NOTCH2-IC) in an EBV-transformed human B lymphoblastoid cell line (LCL) was established. Those genes were compared with the 215 and 154 genes that were, respectively, over- or under-expressed in MZP compared with naive cells (with a fold change ≥1.5, P < 0.05) to generate Venn diagrams. The numbers of genes that are common in both lists are indicated in bold at the intersection between two circles. Hypergeometric distribution was used to calculate the p-value according to GSEA. (E) After gating on dump⁻CD19⁺ cells, and based on their respective expression of MTG, MEM55, CD27, and IgD, naive (gray), MZB (red), MTG⁻MEM55⁺ (blue), and MTG⁺MEM55⁺ (black, dotted line) cells from children spleen samples were analyzed for expression of several surface markers by flow cytometry. Data are representative of 3 different samples.

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