Panel A shows flow cytometry histograms comparing surface expression of CD1d, IgM, CD21, CD23, and IgD on T2, MZ, and FO WT CD45.2 positive B cells from the spleens of WT:WT chimeras. The histograms are color-coded: gold for T2, black for MZ, and blue for FO. Panel B illustrates the gating strategy used to sort CD45.2 positive and CD45.1 positive FO and MZ B cells from the spleens of WT:WT and KO:WT chimeras for RNA sequencing. Panel C presents a volcano plot of differential gene expression between TACI KO and WT CD45.2 positive MZ B cells, showing the mean log2-fold change and the negative log10 adjusted p-value for each gene. Significantly upregulated genes are in red, downregulated genes are in blue, and non-significantly differentially expressed genes are in grey. Panel D shows a similar volcano plot for WT control CD45.1 positive MZ B cells. Panel E depicts a volcano plot for differential gene expression between TACI KO and WT CD45.2 positive FO B cells. Panel F shows flow cytometry histograms comparing pS6-S235 slash 236 levels in resting WT MZ B cells at 37 degrees Celsius with those treated with the mTORC1 inhibitor rapamycin. The histograms are color-coded: black for WT MZ, pink for WT MZ with rapamycin, and grey for isotype control.
Surface marker expression, RNAseq analysis, and pS6 staining. (A) Flow cytometry histograms comparing CD1d, IgM, CD21, CD23, and IgD surface expression on T2 (IgD+IgM+CD93+), MZ (CD1dhiIgMhiCD93−), and FO (CD1dmedIgM+CD93−) WT CD45.2+ B cells (B220+CD19+) from the spleens of WT:WT chimeras. Gold: T2; black: MZ; blue: FO. (B) Gating strategy used to sort CD45.2+ and CD45.1+ FO and MZ B cells from the spleens of WT:WT and KO:WT chimeras by FACS for RNAseq. (C–E) Volcano plot of differential gene expression between TACI KO and WT CD45.2+ MZ B cells (C), between WT control CD45.1+ MZ B cells from KO:WT and WT:WT chimeras (D), and between TACI KO and WT CD45.2+ FO B cells (E) from KO:WT and WT:WT chimeras, respectively, showing the mean log2FC and the −log10padj for each gene from n = 6 mice. Significantly differentially expressed genes are in red (upregulated; log2FC ≥0.263, padj <0.05) and blue (downregulated; log2FC ≤ −0.263, padj <0.05). Gray: not significantly differentially expressed (NS). Dashed gray lines show the thresholds for 1.2-fold change in gene expression and padj = 0.05. (F) Flow cytometry histograms comparing pS6-S235/236 levels in resting WT MZ B cells at 37°C with those treated with the mTORC1 inhibitor rapamycin. Black: WT MZ; pink: WT MZ +10 nM rapamycin; gray: WT MZ cells stained with isotype control antibody. log2FC, log2 fold change; padj, adjusted p-value.