Panel A shows five bar graphs comparing the mean fluorescence intensity (MFI) of CD1d, IgM, CD21, CD23, and IgD on CD45.2 positive and CD45.1 positive splenic marginal zone (MZ) B cells from wild-type (WT) and TACI knockout (KO) mice. The x-axis represents different experimental groups (WT:WT and KO:WT chimeras), and the y-axis represents the MFI normalized to WT CD45.2 positive cells. Panel B displays flow cytometry histograms comparing the surface expression of CD1d, IgM, CD21, CD23, and IgD on TACI KO and WT CD45.2 positive MZ B cells and WT CD45.2 positive follicular (FO) B cells. The x-axis represents fluorescence intensity, and the y-axis represents normalized count. Panel C and D show gene set enrichment analysis (GSEA) plots for the MZ signature (MZ_UP gene set) and the FO signature (FO_UP gene set) in TACI KO versus WT CD45.2 positive splenic MZ B cells. The x-axis represents gene rank, and the y-axis represents enrichment score. Panel E presents bar graphs of mRNA expression levels of Cd1d, Ighm, Cr2, Fcer2a, and Ighd in CD45.2 positive and CD45.1 positive splenic MZ B cells from WT:WT and KO:WT chimeras, quantified as transcripts per million (TPM). The x-axis represents experimental groups, and the y-axis represents TPM.
TACI regulates the MZ B cell transcriptome. (A) CD1d, IgM, CD21, CD23, and IgD surface expression, measured by flow cytometry, on CD45.2+ and CD45.1+ splenic MZ B cells (CD1dhiIgMhiCD93−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 data pooled from two independent analyses. Each point represents one mouse. Black: WT CD45.2+; red: TACI KO CD45.2+; gray: WT CD45.1+. (B) Flow cytometry histograms comparing CD1d, IgM, CD21, CD23, and IgD surface expression on TACI KO and WT CD45.2+ MZ B cells (CD1dhiIgMhiCD93−B220+CD19+) and WT CD45.2+ FO B cells (CD1dmedIgM+CD93−B220+CD19+) from WT:WT and KO:WT chimeras. Histograms are representative of the experiments quantified in A. Red: TACI KO CD45.2+ MZ; black: WT CD45.2+ MZ; blue: WT CD45.2+ FO. (C and D) GSEA of the MZ signature (MZ_UP gene set) (C) and the FO signature (FO_UP gene set) (D) 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 log2 fold change in TACI KO versus WT CD45.2+ MZ B cells (red: upregulated; blue: downregulated; white: no change). The MZ_UP gene set comprises 2,339 genes significantly upregulated ≥1.5-fold in MZ B cells compared with FO B cells, and the FO_UP gene set comprises 1,321 genes significantly upregulated ≥1.5-fold in FO B cells compared with MZ B cells (WT CD45.2+, from this study, Table S2). (E)Cd1d, Ighm, Cr2, Fcer2a, and Ighd mRNA expression in CD45.2+ and CD45.1+ splenic MZ B cells (CD1dhiIgMhiCD93−B220+CD19+) from WT:WT and KO:WT chimeras, determined by RNAseq and quantified as TPM. Bars show the mean. Each point represents one mouse. Black: WT CD45.2+; red: TACI KO CD45.2+; gray: WT CD45.1+. Statistical tests: 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. Sample numbers: 6 (A and E). MFI, mean fluorescence intensity; TPM, transcripts per million; NES, normalized enrichment score; padj, adjusted p-value.