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: 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.

Fig 4: 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.