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. 2019 Nov 14;217(2):e20190860. doi: 10.1084/jem.20190860

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© 2019 du Pré et al.

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Figure 4. — Analysis of 14E06 KI B cells in mixed bone marrow chimeric mice. To create Tgm2^+/+ mixed bone marrow chimeric mice, bone marrow from CD45.2 Tgm2^+/+ 14E06 KI mice was mixed with that from WT CD45.1 mice at a 1:1 ratio to obtain high frequency (HF) bone marrow chimeric mice, or mixed at 1:20 to obtain low frequency (LF) chimeric mice and used to reconstitute the hematopoietic system of lethally irradiated CD45.2 WT recipients. As a control, CD45.2 Tgm2^−/− hosts were reconstituted with bone marrow from CD45.2 Tgm2^−/− 14E06 KI mice and CD45.1 Tgm2^−/−mice mixed 1:1. (A) The frequency of anti-TG2 B cells (as indicated by anti-14E06 staining) in spleens of bone marrow chimeric mice. Data are pooled from two independent measures and presented as mean ± SD from five or six mice in each group. (B) Mixed bone marrow chimeric mice were given BrdU in drinking water for 8 d. Splenic B220⁺CD45.1⁺ (WT) or B220⁺ CD45.2 α14E06⁺ (KI) B cells in Tgm2^−/− control chimeric mice and Tgm2^+/+ HF and Tgm2^+/+ LF chimeric mice were analyzed for BrdU incorporation by flow cytometry. Data are pooled from two independent measures and represent mean ± SD of five or six mice in each group. The BrdU feeding experiment has been performed twice independently, once with LF bone marrow chimeric mice. (C) Immunohistochemical localization of autoreactive TG2-specific B cells in Tgm2^+/+ LF bone marrow chimeric mice. Left: Spleen sections were stained for anti-14E06 (green) to identify anti-TG2 Ig KI B cells, IgM (blue) to identify the B cell zone, and CD169/SIGLEC (magenta) to identify marginal zone macrophages. Right: Spleen sections were stained for CD3 (magenta) to identify the T cell zone in combination with anti-14E06 (green) and B220 (blue). Representative images of two mice (top and bottom panels) out of five mice analyzed are shown. Scale bars, 100 µm. Top panels: 10× objective; bottom panels: 20× objective. (D) Splenic B cells were isolated from Tgm2^−/− (blue), Tgm2^+/+ HF (red), and Tgm2^+/+ LF (green) chimeric mice, loaded with the intracellular calcium indicator Indo-1 and stained with FITC-conjugated anti-CD45.1. Baseline Ca²⁺ levels were acquired for 30 s before the cells were stimulated with 2 µg/ml TG2. CD45.1⁺ cells (WT, dotted lines) and CD45.1-negative cells (comprising mostly 14E06 KI B cells, continuous lines) were gated out and plotted for Ca²⁺ flux analysis. One representative plot out of three independent measures is shown.