Glucocorticoid-inducible natural IgE may be locally produced in mLNs. (A) Level of plasma Cort at ZT4 after 4 wk of Cont (gray) vs. DEX (red) treatment (n = 3 mice/group). (B) Total plasma IgE concentration of Cd4-Cre^+/− GR KO mice after 4 or 5 wk of Cont (gray) vs. DEX (red) treatment. (C) Percentage of CD3e⁺ CD4⁺ T cells (within live CD45⁺ CD11b⁻) in mice injected i.p. with isotype control (gray) vs. anti-CD4 (red) antibody (left; n = 8 mice/group), and total plasma IgE concentration of isotype control vs. CD4-depleted mice after 2 wk of Cont (gray) or DEX (red) treatment (right; n = 4 mice/group). (D) Local IgE levels across various lymphoid tissues from representative IgE KO (row 1) vs. WT B6 (rows 2–7). Numbers on plot and in the legend indicate absolute IgE MFI of FO B cells (CD19⁺ Fas⁻ CD38⁺ IgD⁺), as determined by flow cytometry and described in Fig. 3 D. Dashed vertical line marks peak IgE KO value (representative of six independent experiments). (E) Total plasma IgE concentration (left) and corresponding local IgE levels by absolute FO B cell MFI across various lymphoid tissues (right) for WT mice treated for 4 wk with Cont (gray) vs. DEX (red; n = 4 mice/group). (F) Representative flow cytometric gating for a live CD45⁺ c-Kit⁻ B220⁺ CD19⁺ population, from which true IgE⁺ B cells were quantified from various lymphoid tissues of Cont vs. DEX-treated IgE reporter mice as described in Fig. 3, E and F. Stained samples were validated against their FMO controls for anti-IgE staining (pooled from two to three mice, representative of four independent experiments). (G) Surface protein expression for total or IgE⁺ mLN B cells from Cont (gray) or DEX-treated (red) IgE reporter mice. Numbers indicate MFI by flow cytometry within the appropriate CD19⁺ B220⁺ or CD19⁺ B220⁺ IgE⁺ populations, as described in F. (H) Percentage of true IgE⁺ B cells (within live CD45⁺ c-Kit⁻ CD19⁺ B220⁺) in spleen (left), peritoneal cavity (center), or bone marrow (right) of Cont (gray) vs. DEX-treated (red) IgE reporter mice (n = 2 samples/group with each point representing mean of two to three pooled mice). (I) Multidimensional scaling plot representing relative positions for sorted mLN B cells from Cont vs. DEX-treated mice. x and y axes represent leading log₂(CPM fold change) for DEX/Cont in the first and second dimension, respectively (n = 3 mice/group as biological replicates). (J) Quantification by RNA-seq of mature Ighg1 or Ighm transcripts (top) and upstream γ1GLTs or μGLTs (bottom) of sorted mLN B cells from Cont (gray) vs. DEX-treated (yellow) mice, as represented in Fig. 3, G–I. Numbers on plot indicate CPM for Ighg1 or Ighm (top) or RPKM for γ1GLTs or μGLTs (bottom; n = 3 mice/group). (K) Clonotype abundance for mLN B cells of Cont (left) vs. DEX-treated (right) mice by single cell V(D)J sequencing. Sections represent clonotypes with one (brown), two (yellow), three (orange), or four (red) B cell clones (n = 5,764 B cells pooled from 21 Cont mice or n = 1,449 B cells pooled from 24 DEX-treated mice, compiled from three independent experiments). (L) Fraction of V region genes by number of SHM for mLN B cells of Cont (gray) vs. DEX-treated (red) mice, determined by single-cell V(D)J sequencing (pooled from 21 Cont or 24 DEX-treated mice, compiled from three independent experiments). Error bars in A–C, E, G, H, and L represent SEM. P-values < 0.05 were considered significant (*, P < 0.05; **, P < 0.01; ****, P < 0.0001; N.D., not detected). Welch’s t test was used for A–C and L.