![Figure 9 MZCD9+ B-cell subset is poised for innate immune responses. Heatmap display of NGS data showing enrichment of (A) TLR and (D) innate immune sensors/effector genes. Asterisks denote important genes discussed in the text. qRT-PCR analysis validated NGS results showing MZCD9+ express highest amounts of TLR3, TLR7 and Perforin-2. qRT-PCR (black bars) and RNA-Seq (gray bars) of (B) TLR3, (C) TLR7, and (E) Perforin-2. qRT-PCR data are representative of three independent experiments with FACS-sorted B-cell subsets, where each experiment utilized pooled splenocytes from 2 to 5 mice. (F) qRT-PCR of Perforin-2 mRNA fold-change after stimulation with endosomal TLR agonists. B220 positively selected cells (>95% purity) were stimulated 18 h with either TLR3 agonist Poly(I:C), TLR7/8 agonist CL097, or TLR9 agonist CpG at 1 μg/mL and compared to NS. (G) qRT-PCR of Perforin-2 mRNA fold-change using B220 positive (>95% purity) or CD43 negative (~90% purity) B-cells stimulated with Poly(I:C) as in (F). Data are representative of four independent experiments. (H) qRT-PCR of Perforin-2 mRNA fold-change using FACS derived MZCD9+ or FO-I B-cells treated with Poly(I:C) as in (F). *P ≤ 0.05; **P ≤ 0.01.](https://i0.wp.com/idsc.miami.edu/wp-content/uploads/2020/10/fimmu-06-00030-g009.jpg?resize=940%2C530&ssl=1)
Distinct Transcriptomic Features and B-Cell Populations in the Mouse…
Splenic transitional B-cells (T1 and T2) are selected to avoid self-reactivity and to safeguard against autoimmunity, then differentiate into mature follicular (FO-I and FO-II) and marginal zone (MZ) subsets. Transcriptomic analysis by RNA-seq of the five B-cell subsets revealed T1 cell signature genes included RAG suggesting a potential for receptor revision. T1 to T2 B-cell differentiation was marked by a switch from Myb to Myc, increased expression of the PI3K adapter DAP10 and MHC class II. FO-II may be an intermediate in FO-I differentiation and may also become MZ B-cells as suggested by principle component analysis. MZ B-cells possessed the most distinct transcriptome including down-regulation of CD45 phosphatase-associated protein (CD45-AP/PTPRC-AP), as well as upregulation of IL-9R and innate molecules TLR3, TLR7, and bactericidal Perforin-2 (MPEG1). Among the endosomal TLRs, stimulation via TLR3 further enhanced Perforin-2 expression exclusively in MZ B-cells. Using gene-deleted and overexpressing transgenic mice we show that IL-9/IL-9R interaction resulted in rapid activation of STAT1, 3, and 5, primarily in MZ B-cells. Importantly, CD45-AP mutant mice had reduced transitional and increased mature MZ and FO B-cells, suggesting that it prevents premature entry of transitional B-cells to the mature B-cell pool or their survival and proliferation. Together, these findings suggest, developmental plasticity among splenic B-cell subsets, potential for receptor revision in peripheral tolerance whereas enhanced metabolism coincides with T2 to mature B-cell differentiation. Further, unique core transcriptional signatures in MZ B-cells may control their innate features.
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Distinct Transcriptomic Features are Associated with Transitional and Mature B-Cell Populations in the Mouse Spleen by Eden Kleiman, Daria Salyakina, Magali De Heusch, Kristen L. Hoek, Joan M. Llanes, Iris Castro, Jacqueline A. Wright, Emily S. Clark, Derek M. Dykxhoorn, Enrico Capobianco, Akiko Takeda, Jean-Christophe Renauld, and Wasif N. Khan. PMCID: PMC4324157