Volume 18, Issue 7, Pages 1627-1635 (February 2017) BCR and Endosomal TLR Signals Synergize to Increase AID Expression and Establish Central B Cell Tolerance  Masayuki Kuraoka, Pilar B. Snowden, Takuya Nojima, Laurent Verkoczy, Barton F. Haynes, Daisuke Kitamura, Garnett Kelsoe  Cell Reports  Volume 18, Issue 7, Pages 1627-1635 (February 2017) DOI: 10.1016/j.celrep.2017.01.050 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 18, 1627-1635DOI: (10.1016/j.celrep.2017.01.050) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Anti-μ+CpG Co-activation Synergistically Elevated AID mRNA Expression in Immature/T1 B Cells (A and B) qPCR analysis of AID mRNA levels in bone marrow immature/T1 B cells (A) and splenic MF B cells (B) cultured for 24 hr in the presence of indicated stimuli (n = 4–15). AID expression in splenic GC B cells (▿; n = 4) from chicken gamma globulin conjugated with (4-hydroxy-3-nitrophenyl)acetyl (NP) in alum (NP-CGG/alum) immunized mice are shown in both panels. Each point represents an individual mouse and determination from at least four independent experiments. NS, not significant, p > 0.05; ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, unpaired Student’s t test. See also Figure S4. Cell Reports 2017 18, 1627-1635DOI: (10.1016/j.celrep.2017.01.050) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 PLD Activation, Endosomal Acidification, and Myd88 Are Required for Anti-μ+CpG-Induced Synergistic AID Upregulation in Immature/T1 B Cells (A–D) Representative images of immature/T1 B cells (IgM, TLR9, differential interference contrast [DIC], and merged images) cultured with CpG (A), anti-μ+CpG (B), anti-μ+CpG+n-butanol (C), and anti-μ+CpG+chloroquine (D). Top and bottom represent two independent cells. Scale bars, 5 μm. (E–G) AID mRNA levels in immature/T1 B cells stimulated with CpG or anti-μ+CpG in the presence of various concentrations of (E) n-butanol (v/v, n = 4) or (F) chloroquine (n = 3–4). (G) AID mRNA levels in immature/T1 B cells from B6 and B6.Myd88−/− mice before (n = 13) and after culture (n = 4) in the presence of CpG or anti-μ+CpG. Each point represents an individual mouse and determination from at least two independent experiments. NS, not significant, p > 0.05; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, unpaired Student’s t test. See also Figure S1. Cell Reports 2017 18, 1627-1635DOI: (10.1016/j.celrep.2017.01.050) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Autoreactive Immature/T1 B Cells Are Not Efficiently Purged in the Absence of Myd88 (A–D) Single immature/T1 B cells isolated from indicated mice were grown in Nojima cultures (A) and (D). Culture supernatants containing 1 to 3 μg/mL IgG were screened for DNA reactivity by ELISA. Anti-DNA IgG AvIn of individual cultures of immature/T1 B cells (B6, n = 45; B6.Myd88−/−, n = 43; 3H9, n = 63; 3H9.Myd88−/−, n = 64) are shown. Each point represents an individual immature/T1 B cell culture and determination from at least two independent experiments. Boxes extend from the 25th to 75th percentiles, and lines in the boxes represent median. Error bars represent the 10th to 90th percentiles. ∗∗p < 0.01, ∗∗∗∗p < 0.0001, Mann-Whitney’s U test. (B) Representative flow diagrams for IgM/IgD expression by bone marrow cells of B1-8i, 3H9, and 3H9.Myd88−/− mice. Numbers near boxes represent frequencies of immature B cells (imm; IgMloIgD−), T1 B cells (T1; IgMhiIgD+/−) and mature B cells (mature; IgMintIgDhi). (C) Absolute cell numbers of immature/T1 B cells in bone marrow of B1-8i (n = 9), 3H9 (n = 29), and 3H9.Myd88−/− (n = 12) mice. NS, not significant, p > 0.05; ∗∗∗∗p < 0.0001, unpaired Student’s t test; error bars, SEM. (E) Number of immature/T1 B cells (relative to 3H9) in bone marrow of B1-8i (n = 9), 3H9 (n = 29), 3H9.Aicda+/− (n = 5), 3H9.Myd88+/− (n = 7), 3H9.Aicda+/−Myd88+/− (n = 18), 3H9.Aicda−/− (n = 10), and 3H9.Myd88−/− (n = 12) mice. Error bars, SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, unpaired Student’s t test. See also Figure S2. Cell Reports 2017 18, 1627-1635DOI: (10.1016/j.celrep.2017.01.050) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Endosomal Acidification Is Required for Central B Cell Tolerance (A and B) Representative flow plots for IgM/IgD expression by B220loCD93+CD43−CD23− small bone marrow lymphocytes (A), and ratio of immature/T1 B cells to small pre-B cells (B) in B1-8 mice (n = 9), and PBS-treated (n = 7) or chloroquine-treated (n = 10) 3H9 mice from five independent experiments. (C and D) Single immature/T1 B cells isolated from PBS- or chloroquine-treated 3H9 mice were grown in Nojima cultures (see legend for Figures 3A and 3D). (C) Anti-DNA IgG AvIn of individual immature/T1 B cell cultures [n = 206 and 186 for PBS-treated (n = 5) and chloroquine-treated (n = 5) 3H9 mice, respectively]. Boxes extend from the 25th to 75th percentiles, and lines in the boxes represent median. Error bars represent the 10th to 90th percentiles. (D) Anti-DNA IgG AvIn of immature/T1 B cell cultures of B6 (Figure 2A, gray), 3H9 (C; PBS-treated, green; chloroquine-treated, blue), and 3H9.Myd88−/− mice (Figure 3D; purple) were compartmentalized by binning into 2-fold intervals. (E) Ratio of indicated B cell compartments to small pre-B cells compared between control (n = 5) and chloroquine-treated (n = 7) 2F5 dKI mice. Boxes extend from the 25th to 75th percentiles, and lines in the boxes represent median. Bars represent the range (minimum/maximum) of values. Each symbol represents individual mice from two independent experiments. ∗p < 0.05, ∗∗∗p < 0.001, Mann-Whitney’s U test. Error bars, SEM. See also Figure S3. Cell Reports 2017 18, 1627-1635DOI: (10.1016/j.celrep.2017.01.050) Copyright © 2017 The Author(s) Terms and Conditions