Supplementary Figure 6: Related to Fig. 6. Ikaros-regulated genes in anergic B cells.

a, Flow cytometric sorting of splenic FO B cells as exemplified for MD4 BCR-tg ML5-tg Ikzf1^B+ FO B cells. After immunomagnetic depletion with CD43-MicroBeads, FO B cells were sorted as CD19⁺CD23⁺CD21^int CD93^int/–TCRβ^–CD138^– cells. Reanalysis determined a purity of > 95% (here 98.5%) for the sorted live FO B cells. b, Absence of exon 8 in Ikzf1 transcripts following gene inactivation. The combined RNA-seq profile of all replicate experiments is shown for naïve and 1-day HEL-exposed FO B cells of the indicated genotypes. The floxed exon 8 is marked by gray shading. c,d, Scatter plot of gene expression differences between naïve FO B cells of the control MD4 BCR-tg Ikzf1^B+ and Ikaros mutant MD4 BCR-tg Ikzf1^B– genotypes (c) as well as between 1-day HEL-exposed FO B cells of same control and Ikaros mutant genotypes (d). The expression of each gene was plotted as normalized log₁₀ (norm rlog) expression value (see Online Methods). Ikaros-regulated genes were defined by an expression difference of > 2-fold, an adjusted P value of < 0.05 and a TPM (transcripts per million) value of > 5 in B cells of the control or Ikaros mutant genotype, respectively, which resulted in the identification of the indicated number of Ikaros-activated (blue) and Ikaros-repressed (red) genes in naïve FO B cells (c; Table 1) or HEL-exposed FO B cells (d; Table 2). The dot size varies according to the adjusted P value calculated for each gene. e, Overlap of the Ikaros-dependent gene expression patterns between naïve and 1-day HEL-exposed FO B cells (Table 3). The log₂-fold expression change observed between HEL-exposed Ikaros-deficient (MD4 BCR-tg Ikzf1^B–) and control (MD4 BCR-tg Ikzf1^B+) FO B cells (horizontal axis) as well as between naive Ikaros-deficient and control FO B cells (vertical axis) is plotted for each gene. Differentially expressed genes, which passed the above mentioned selection criteria (c,d), are marked by dark blue dots (Ikaros-activated) or dark red dots (Ikaros-repressed) in the naïve FO B cell comparison or by blue open circles (Ikaros-activated) and red open circles (Ikaros-repressed) in the comparison of the HEL-exposed FO B cells. The size of the circles varies according to the adjusted P value calculated for each gene in HEL-exposed FO B cells. A Pearson coefficient of 0.67 and a regression slope of 0.53 were calculated for the comparison of the Ikaros-dependent gene expression patterns between naïve and HEL-induced FO B cells. Based on the above mentioned selection criteria (c,d), 42 Ikaros-repressed and 15 Ikaros-activated genes overlapped in naïve and HEL-treated FO B cells (closed circles). f, Functional classification and quantification of the proteins that are encoded by Ikaros-activated and Ikaros-repressed genes of the Ikaros-dependent gene expression signature (Fig. 6a, Table 3). The bar size indicates the percentage of activated or repressed genes in each functional class relative to the total activated or repressed genes, respectively. Numbers in the bars indicate the number of genes in each functional class. g, Definition of an RNA-seq-based anergy expression signature (volcano plot, lower part) and its correlation with a previously published anergy expression signature (density profile, upper part). Based on RNA-seq analyses of naïve MD4 BCR-tg FO B cells and fully anergic MD4 BCR-tg ML5-tg FO B cells, an anergy expression signature was defined by selecting genes with an expression difference of > 3-fold, an adjusted P value of < 0.05 and a TPM value of > 5 in naïve or anergic FO B cells, respectively, which identified 59 genes (green dots) with increased expression (‘anergy up’) and 50 genes (brown dots) with decreased expression (‘anergy down’) in anergic FO B cells compared to naïve B cells (Table 4). Black circles denote genes, which are described as ‘strongly’ regulated genes in the anergy expression signature of Sabouri et al. (Nat. Commun. 7, 13381). A density profile (upper part) shows the correlation of the published ‘strongly’ and ‘moderately-to-strongly’ regulated anergy signature genes with the log₂-fold expression changes of the differentially expressed genes defined by the RNA-seq comparison of naïve and anergic FO B cells. h, Gene set enrichment analysis (GSEA) of the 59 ‘anergy up’ genes (upper panel) and 50 ‘anergy down’ genes (lower panel) based on the ranked log₂-fold gene expression changes determined for Ikaros-deficient versus control HEL-exposed FO B cells. NES, normalized enrichment score; FDR, false discovery rate. i, Expression of Ighm and Ighd mRNAs (coding for IgM and IgD) in naïve or HEL-exposed Ikaros-deficient FO B cells (dark purple) and in naïve or HEL-exposed control FO B cells (light purple). Gray bars display Ighm and Ighd expression in fully anergic MD4 BCR-tg ML5-tg FO B cells, while the light purple bar overlaid on the gray bar indicates the corresponding expression in naïve MD4 BCR-tg FO B cells. Data in a are representative of the sorting of FO B cells, used for RNA-seq analysis. The RNA-seq data shown in b-i were obtained from 2 (naïve FO B) and 5 (HEL-exposed FO B) independent experiments. Statistical data (i) are shown as mean values with SEM.