Figure 3.
Natural IgE is produced in mLNs by glucocorticoids in vivo. (A) Total plasma IgE concentration of WT mice treated with Cont (gray) vs. DEX (red) in drinking water for 2 or 4 wk, as represented in the experimental setup (n = 29 mice/group, compiled across five independent experiments). (B) Total plasma IgG1, IgG2a, IgG3, IgA, IgM, or IgG2b concentration of WT mice after 4 wk of Cont (gray) vs. DEX (red) treatment (n = 6 mice/group, compiled across two independent experiments). (C) Total plasma IgE concentration of Mb1-Cre+/− GR WT or Het (gray) vs. cKO (red) mice at steady state (0 wk), or after 2 or 4 wk of DEX treatment (n = 5–6 mice/group). (D) Relative levels of local IgE across various lymphoid tissues of B6 (red) or IgE KO (gray) mice, assessed by MFI of IgE bound to CD23 of FO B cells (CD19+ Fas− CD38+ IgD+) as a proxy for local IgE concentration in the tissue. Numbers indicate relative IgE MFI, normalized to MFI of the corresponding inguinal LN and determined by flow cytometry (n = 1–12 mice/group, compiled from six independent experiments). (E) Percentage of true mLN IgE+ B cells (within live CD45+ c-Kit− CD19+ B220+ population) of IgE reporter mice treated for 4 wk with Cont (gray) vs. DEX (red), as represented in the experimental setup (n = 8 samples/group with each point representing mean of two to three pooled mice, compiled from four independent experiments). (F) Representative flow cytometric gating for IgE+ B cells in mLNs of Cont (left) vs. DEX-treated (right) IgE reporter mice. True IgE-expressing cells were identified as double positive for fluorescent Venus (x axis) and anti-IgE stain (y axis) within live CD45+ c-Kit− B220+ CD19+ population, as described in E (pooled from two to three mice, representative of four independent experiments). (G) Quantification by RNA-seq of mature Ighe transcripts and upstream εGLTs of sorted mLN B cells from mice treated for 4 wk with Cont (gray) or DEX (yellow). Numbers indicate CPM for Ighe (top) or RPKM from upstream regions of Ighe gene for εGLTs (bottom; n = 3 mice/group). (H) Hierarchical clustering by and relative expression of genes associated with plasmablast-like or germinal center and memory B cell–like signatures for sorted mLN B cells from Cont vs. DEX-treated mice. Color gradations indicate z-scores by CPM (n = 3 mice/group as biological replicates). (I) Percentage of IgE+ IgG1− B cells (within live B220+) from splenic vs. mLN WT CD19+ B cells cultured with Cont (gray) or DEX (red), as described in Fig. 1 C (n = 3 mice/group). Error bars in A–D and G represent SEM. P values <0.05 were considered significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant). Welch’s t test was used for A–C and E, and ratio paired Student’s t test was used for D.

Natural IgE is produced in mLNs by glucocorticoids in vivo. (A) Total plasma IgE concentration of WT mice treated with Cont (gray) vs. DEX (red) in drinking water for 2 or 4 wk, as represented in the experimental setup (n = 29 mice/group, compiled across five independent experiments). (B) Total plasma IgG1, IgG2a, IgG3, IgA, IgM, or IgG2b concentration of WT mice after 4 wk of Cont (gray) vs. DEX (red) treatment (n = 6 mice/group, compiled across two independent experiments). (C) Total plasma IgE concentration of Mb1-Cre+/− GR WT or Het (gray) vs. cKO (red) mice at steady state (0 wk), or after 2 or 4 wk of DEX treatment (n = 5–6 mice/group). (D) Relative levels of local IgE across various lymphoid tissues of B6 (red) or IgE KO (gray) mice, assessed by MFI of IgE bound to CD23 of FO B cells (CD19+ Fas− CD38+ IgD+) as a proxy for local IgE concentration in the tissue. Numbers indicate relative IgE MFI, normalized to MFI of the corresponding inguinal LN and determined by flow cytometry (n = 1–12 mice/group, compiled from six independent experiments). (E) Percentage of true mLN IgE+ B cells (within live CD45+ c-Kit− CD19+ B220+ population) of IgE reporter mice treated for 4 wk with Cont (gray) vs. DEX (red), as represented in the experimental setup (n = 8 samples/group with each point representing mean of two to three pooled mice, compiled from four independent experiments). (F) Representative flow cytometric gating for IgE+ B cells in mLNs of Cont (left) vs. DEX-treated (right) IgE reporter mice. True IgE-expressing cells were identified as double positive for fluorescent Venus (x axis) and anti-IgE stain (y axis) within live CD45+ c-Kit− B220+ CD19+ population, as described in E (pooled from two to three mice, representative of four independent experiments). (G) Quantification by RNA-seq of mature Ighe transcripts and upstream εGLTs of sorted mLN B cells from mice treated for 4 wk with Cont (gray) or DEX (yellow). Numbers indicate CPM for Ighe (top) or RPKM from upstream regions of Ighe gene for εGLTs (bottom; n = 3 mice/group). (H) Hierarchical clustering by and relative expression of genes associated with plasmablast-like or germinal center and memory B cell–like signatures for sorted mLN B cells from Cont vs. DEX-treated mice. Color gradations indicate z-scores by CPM (n = 3 mice/group as biological replicates). (I) Percentage of IgE+ IgG1− B cells (within live B220+) from splenic vs. mLN WT CD19+ B cells cultured with Cont (gray) or DEX (red), as described in Fig. 1 C (n = 3 mice/group). Error bars in A–D and G represent SEM. P values <0.05 were considered significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant). Welch’s t test was used for A–C and E, and ratio paired Student’s t test was used for D.

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