Transcriptomics Identify CD9 as a Marker of Murine IL-10-Competent Regulatory B Cells - PubMed (original) (raw)

Transcriptomics Identify CD9 as a Marker of Murine IL-10-Competent Regulatory B Cells

Jianbo Sun et al. Cell Rep. 2015.

Abstract

Regulatory B cells (Breg) have immune suppressive functions in various autoimmune/inflammation models and diseases and are found to be enriched in diverse B cell subsets. The lack of a unique marker or set of markers efficiently identifying Breg cells impedes detailed investigation into their origin, development, and immunological roles. Here, we perform transcriptome analysis of IL-10-expressing B cells to identify key regulators for Breg biogenesis and function and identify CD9, a tetraspanin-family transmembrane protein, as a key surface marker for most mouse IL-10(+) B cells and their progenitors. CD9 plays a role in the suppressive function of IL-10(+) B cells in ex vivo T cell proliferation assays through a mechanism that is dependent upon B/T cell interactions. CD9(+) B cells also demonstrate inhibition of Th1-mediated contact hypersensitivity in an in vivo model system. Taken together, our findings implicate CD9 in the immunosuppressive activity of regulatory B cells.

Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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Conflict of interest statement

Competing financial interests: The authors declare they have no competing financial interests.

Figures

Figure 1

Figure 1. Differentially expressed mRNA, lncRNA and miRNA in B10 cells

(A) Overall changes of gene expression level between B10+ cells (CD1dhiCD5+CD19+IL10+) and B10− cells (CD1d−CD5−CD19+IL10−) determined by RNA-seq. *: the number of mRNAs after removing mRNAs with FPKM<1 in both B10+ and B10− cells in any batch, and mRNA with FPKM=0 in any sample. See Supplemental Experimental Procedures for more information. (B) The distribution of genes in the top iFC and iRC groups. The gene list is summarized in Table S1. (C) The rank changes of the 55 genes existing in both the top 100 iFC and the top 100 iRC. IL10 is ranked first in both rank lists. (D) Fold change of the 44 novel lncRNAs identified from the lncRNA database (Pefanis et al., 2014; Pefanis et al., 2015). The lncRNAs are nomenclatured according to the gene closest to the lncRNA coding region. (E) General changes of miRNA expression levels between B10+ cells and B10− cells as determined by microarray analysis. “Max signal”: the maximum signal in all 4 samples of the two batches. (F) The expression changes of the miRNAs with iFC≥ 3 and max signal ≥ 32. Please also see Figure S1 and S2, Tables S1, S2, and S3.

Figure 2

Figure 2. IL10competent B cells and their progenitors are distinguished by CD9

(A–D) Screening markers for IL10competent B cells identified by RNA-seq. (A) The mapped reads of differentially expressed CD antigens as well as CCR1 determined by RNA-seq on B10+ and B10− cells. (B and C) Representative expression of IL10 and the molecules by total B cells (the dot plots), and expression of the molecules on CD19+IL10+ and CD19+IL10− cells (the histogram plots) determined by flow cytometry. The percent population is shown for CD19+IL10+ cells. All B cells are enriched with CD19 micro-beads and then treated with LPI+M for 5h (C) or CD40 antibody for 48h plus LPI+M for the terminal 5h (B). (D) Statistical results of expression of various molecules on IL10+ or IL10− B cells. (E) Expression of IL10 and CD9 on CD19+ B cells isolated from peritoneal cavity (PC) B cells or splenic (SP) B cells and treated with LPI+M for 5h, or CD40 antibody for 48h followed by LPI+M for the terminal 5h. Two different CD9 antibodies were used. The bar graph shows the statistical frequency of subsets in total or IL10+ B cells. (F) Expression change of IL10 and CD9 on the indicated sorted cell subsets after incubation with CD40 antibody for 48h followed by LPI+M for the terminal 5h. The bar graph shows the statistical frequency of IL10+ in the sorted subsets. Cell subsets were sorted from naive B cells treated with (for IL10 staining) or without (for CD19 and CD9 staining only) LPI for 5h. Statistical data are represented as mean with SEM. p-value was obtained by multiple t-test. Please also see Figure S3 and Table S4.

Figure 3

Figure 3. CD9 is a robust surface marker for mouse IL10competent B cells

(A) Representative expression of IL10, CD9 and other reported Breg markers on total, IL10+ or IL10− B cells stimulated with LPI+M for 5h or CD40 antibody for 48h plus LPI+M for the terminal 5h. The frequency of cells expressing specific markers on IL10+ B cells is shown in the histograms. (B) The statistical frequency of subsets expressing specific markers on IL10− or IL10+ B cells. Statistical data are represented as mean with SEM.

Figure 4

Figure 4. CD9 is involved in B10 function and biogenesis

(A) Representative and statistical proliferation of T cells co-cultured for 48h and 72h at the indicated initial ratio with CD9+ or CD9− B cells. The statistical results are normalized by the T cells co-cultured without B cells. (B) Representative and statistical proliferation of T cells co-cultured for 72h at the indicated initial ratio with CD9+ or CD9− B cells in the presence of antibodies. (C) Representative and statistical proliferation of T cells (100×103) in the insert or in the receiver well of the transwell plate. The insert was added at the indicated initial ratio with CD9+ or CD9− B cells as shown in the bottom cartoon and incubated for 72h. (D) Statistical analysis of IL10 production by CD9+ B cells and CD9− B cells treated with PMA, ionomycin and monensin for the terminal 5h of culture. (A-D) CD9+ or CD9− B cells were sorted from CD19+ B cells treated with CD40 antibody and LPS for 5h. CD4+CD25− T cells were sorted from naive CD19− cells and then stained by VPD450 before culturing in the plates pretreated with 5ug/ml CD3e antibody. Medium was changed at 48h. (E) Ear thickness change of CD19 knockout mice adoptively transferred with CD9+ B cells or CD9− B cells in an in vivo B cell functional analysis assay. WT and CD19 KO mice injected with PBS were the controls for the experiment. The cells were sorted as described in Materials and Methods. (F) The frequency of MZ B and FO B in total B cells or IL10+ B cells, and the frequency of IL10+ B cells in total, FO, and MZ B cells. Statistical data are represented as mean with SEM. P-values were obtained by multiple t-test. Please also see Figure S4.

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