Fig 1: RELMα Expression by B Cells Is Required for Limiting Nb-Induced Emphysematous Pathology(A) B cell numbers in the lung at different time points after N. brasiliensis inoculation were assessed by flow cytometry.(B) At day 5 after Nb inoculation, sort-purified B cells were analyzed for mRNA expression of candidate regulatory factors.(C) Sort-purified B cells from Il4ra−/− and WT BALB/c mice were analyzed for Retnla mRNA expression at day 5 after Nb inoculation.(D–F) Sort-purified B cells (D), alveolar macrophages (E), or non-alveolar macrophages (F) at different time points after Nb infection were analyzed for Retnla mRNA expression.(G and H) RELMα mean fluorescence intensity (MFI) of lung B cells (CD19+) (G) and alveolar macrophages (H) was determined by cytoplasmic staining and FACS at day 5 after N. brasiliensis inoculation.(I and J) Sort-purified B cells from either untreated Retnla−/− or WT mice were transferred to recipient Jh−/− mice at days −3, 0, and +1 after N. brasiliensis inoculation. Lungs were collected for analysis 7 days after inoculation, emphysematous pathology was digitally imaged as described in Figure 1 (I), and lung tissues were analyzed for the expression of Il17a by qPCR (J).(K) Retnla−/− and WT mice were inoculated with Nb, and 2 days later, γδ T cells were assessed for IL-17A production by intracellular staining and flow cytometric analyses.(L and M) FACS analysis of lung B cell subsets at day 5 after N. brasiliensis inoculation, showing expression of IgM and IgD (L) and CD21, CD23, and CD24 (M) on gated CD19+ lymphocytes.Each symbol represents an individual mouse. Small horizontal lines indicate the mean (A and D–I), or data shown are the mean and SEM from five individual mice per group (B–F and J) or a pool of five mice per group (K and L). Data shown are representative of at least two independent experiments (**p < 0.01). See also Figures S4 and S5.
Fig 2: GPR182-/- mice have a reduced marginal zone. (A) Representative FACS plots indicating the percentage of MZBs (CD21Hi CD23- cells gated on CD19+ B220+) in GPR182+/- (left) and GPR182-/- (right) mice. (B) Quantification of multiple measurements, means ± SD (N = at least 7/group). (C) Representative confocal images of CD21/35 staining (green) to show MZ, found at the border with the marginal sinus, MAdCAM-1 (red) (left, scale bar = 200μm). Right panels: enlargements with measurement bar used for quantification (right, scale bar = 50μm). (D) Quantification of MZ thickness (N=5 spleens/group, with at least 5 follicles/spleen analyzed). Mean fluorescence intensity (MFI) of receptor expression on MZBs CXCR4 (E) and CXCR5 (F); and on FoBs CXCR4 (G) and CXCR5 (H), CD29 expression (integrin β1 chain) on MZB (I) (N= at least 7 mice/group). (J) Representative confocal images of deposition of TNP-Ficoll-fluorescein (green) on spleen FDCs (CD21, red) within the follicle (lined by MAdCAM-1+ marginal sinus, cyan), in GPR182+/- (left) and GPR182-/- (right) mice, 3h post-injection. (K) Quantification of TNP-Ficoll-fluorescein deposition on CD21+ FDCs of GPR182+/- (black circles) and GPR182-/- (red circles) mice (N=5 mice/group). (L) Levels of TNP-specific IgM at day 4 post immunization in serum of GPR182+/- (black circles) and GPR182-/- (red circles) mice measured by ELISA (N=7 mice/group). Error bars means ± SD, mean thick horizontal line. Unpaired two-tailed t-test: *p<0.05, ***p<0.001, ****p<0.0001, ns not significant).
Fig 3: SMF exposure has little effect on peripheral B cell proliferation, apoptosis and the expression of CD19, IgD, CD23, and CD21 on B cells. (A–D) MFI of CD19, IgD, CD23, and CD21 in splenic B cells from sham and 33.0 T/28.7 T/17.8 T/11.2 T SMF‐exposed groups. (E and F) Percentage of Annexin V and Ki67‐positive cells in total splenic B cell and FO, MZ, T1, T2, B subsets in sham and 33.0 T SMF‐exposed groups. (G–I) Percentage of Annexin V positive cells in total splenic B cell and FO, MZ, T1, T2, B subsets in sham and 28.7 T/17.8 T/11.2 T SMF‐exposed groups. (J and K) Percentage of Ki67‐positive cells in total splenic B cell and FO, MZ, T1, T2, B subsets in sham and 28.7 T/17.8 T SMF‐exposed groups. Samples were analyzed with FlowJo software, Error bars were shown as mean ± SEM. Dots represent individual mouse. *p < 0.05; **p < 0.01; ***p < 0.001 and ns: no statistical significance.
Fig 4: Splenic distribution of B-, T- and NK-cell subpopulations. (A) A representative bivariate density plot of CD21 (y-axis) vs CD23 (x-axis) for the analysis of the compartmental distribution of B cells in the spleen of mice with and without 4-NQO exposure at 8 (i) and 24 (ii) weeks using FlowJo Software v8.0.2. Mean and standard error of the mean of percent total of (B) CD11b−/CD5−/CD19+/CD21+Mid/CD23+ follicular B cells (C) CD11b−/CD5−/CD19+/CD21+hi/CD23− marginal zone B cells and (D) CD11b−/CD5−/CD19+/CD21−/CD23– immature B and B1a cells. (E) A representative bivariate density plot of CD43 (y-axis) vs γδTCR (x-axis) for the analysis of the compartmental distribution of T cells in the spleen of mice with and without 4-NQO exposure at 8 (i) and 24 (ii) weeks using FlowJo Software v8.0.2. Mean and standard error of the mean of percent total of (F) CD11b−/CD19−/CD5+/CD43+/γδ-TCR+ gamma/delta-T cells and (G) CD11b−/CD19−/CD5+/CD43+/γδ-TCR− alpha/beta-T cells. (H) A representative bivariate density plot of CD161 (y-axis) vs CD43 (x-axis) for the analysis of the compartmental distribution of NK cells in the spleen of mice with and without 4-NQO exposure at 8 (i) and 24 (ii) weeks using FlowJo Software v8.0.2. Mean and standard error of the mean of percent total of (I) CD11b−/CD5−/CD19−/CD161+/CD43− NK cells and (J), CD11b−/CD5−/CD19−/CD161+/CD43+ NK cells. Mean ± SD, ns, non-significant, *P-value <0.05, **P-value <0.01, and ***P-value <0.001.
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