Increased FOXO1 activity in TACI-deficient MZ B cells. (A) Volcano plot of gene set enrichment in TACI KO versus WT CD45.2⁺ MZ B cells for 475 TF target gene sets, showing the NES and −log₁₀padj for each gene set determined by GSEA. Significantly differentially expressed TF target gene sets are in red (upregulated; NES ≥1, padj <0.05) and blue (downregulated; NES ≤−1, padj <0.05). Gray: not significantly differentially expressed (NS). Results for FOXO1, FOXO3, and NOTCH1 target gene sets are shown. (B) GSEA of FOXO1-activated target genes in TACI KO versus WT CD45.2⁺ splenic MZ B cells. The plot shows the running enrichment score (green line) above the ranked list of genes, ordered from highest to lowest log₂FC in TACI KO versus WT CD45.2⁺ MZ B cells (red: upregulated; blue: downregulated; white: no change). The “FOXO1_activated target genes” gene set comprises 158 genes that are bound by FOXO1 in mouse lymphocytes and are downregulated following FOXO1 KO, curated as described in Materials and methods. (C) Volcano plot of differential gene expression between TACI KO and WT CD45.2⁺ MZ B cells from KO:WT and WT:WT chimeras, respectively, with differentially expressed FOXO1-activated or FOXO1-repressed target genes highlighted (orange: FOXO1-activated and upregulated, log₂FC ≥0.263, padj <0.05; blue: FOXO1-activated and downregulated, log₂FC ≤ −0.263, padj <0.05; purple: FOXO1-repressed and downregulated). The mean log₂FC in KO versus WT cells and −log₁₀padj are shown for each gene from n = 6 mice. (D)Cxcr4, Bach2, Klf2, Ccr7, Cd55, and Prdm1 mRNA expression in CD45.2⁺ and CD45.1⁺ splenic MZ B cells (CD1d^hiIgM^hiCD93⁻B220⁺CD19⁺) from WT:WT and KO:WT chimeras, determined by RNAseq and quantified as TPM. Bars show the mean of n = 6 mice. Each point represents one mouse. Black: WT CD45.2⁺; red: TACI KO CD45.2⁺; gray: WT CD45.1⁺. (E) CD55 and CXCR4 surface expression, measured by flow cytometry, on CD45.2⁺ and CD45.1⁺ splenic MZ B cells (CD1d^hiIgM^hiCD93⁻B220⁺CD19⁺) from WT:WT and KO:WT chimeras, quantified as the geometric MFI and normalized to the mean MFI of WT CD45.2⁺ cells within the same experiment. Bars show the mean of n = 6 (WT:WT) and n = 7 (KO:WT) mice, pooled from two independent analyses. Each point represents one mouse. Black: WT CD45.2⁺; red: TACI KO CD45.2⁺; gray: WT CD45.1⁺. Statistical tests in D and E: repeated-measures two-way ANOVA with Fisher’s LSD test. Numbers above graphs indicate P values where P ≤ 0.05, otherwise no value is shown. (F and G) GSEA of AKT-positively regulated genes (AKT^BOE_UP gene set) (F) and of AKT-negatively regulated genes (AKT^BOE_DOWN gene set) (G) in TACI KO versus WT CD45.2⁺ splenic MZ B cells, displayed as in B. The AKT^BOE_UP gene set comprises 1,478 genes that are upregulated, and the AKT^BOE_DOWN gene set comprises 909 genes that are downregulated in B cells overexpressing constitutively activated AKT1 (AKT^BOE), compared with control B cells. MFI, mean fluorescence intensity; TPM, transcripts per million; log₂FC, log₂ fold change; NES, normalized enrichment score; padj, adjusted p-value.