Figure 6: FALC B1-cell activation requires IL-5 but does not require eosinophils. | Nature Communications

Figure 6: FALC B1-cell activation requires IL-5 but does not require eosinophils.

From: Fat-associated lymphoid clusters control local IgM secretion during pleural infection and lung inflammation

Figure 6

(a) Schematic of the i.pl. transfer experiment of CTV-labelled Il1rl1−/− and CFSE-labelled BALB/c control PLEC into BALB/c recipient mice before Alt treatment. (b) Flow cytometric analysis of digested pericardial and mediastinal FALCs showing gating of CFSE and CTV-labelled B1 cells (gated as CD45+CD19+CD11b+). CFSE and CTV intensities (upper histograms) and Ki67 expression (lower histogram) are shown for CFSE (left) and for CTV-labelled B1 cells (right). Quantification of %Ki67 expression within CFSE+ BALB/c and CTV+ Illrl1−/− B1-cell populations. Data combined from two independent experiments, symbols represent individual recipient mice, n=4. (c–h) Schematic of αIL-5 i.pl. blocking experiment with mice that received: intra-nasal (i.n.) and intra-pleural PBS, i.n. Alt and intra-pleural IgG control, i.n. Alt and intra-pleural anti-IL-5 blocking IgG (c). Flow cytometric analysis of digested pericardial FALCs, %Ki67high B1a and B1b cells in pericardial FALCs (d). Representative whole-mount immunofluorescence staining of mediastinal FALCs showing IgM (green), Ki67 (red) and CD45 (blue) (e). Scale bar, 50 μm. Quantification of % area of Ki67 staining (f). ELISA of total IgM secreted during 4 h of in vitro culture of whole mediastina (g). Quantification of eosinophil numbers within PLEC and digested pericardium (h). Data in d–h combined from 3 (d–g) and 2 (h) independent experiments, symbols represent individual mice, n= 13 or 15 (d–g) and n=5 or 10 (h) per group. (i–k) BALB/c and ΔdblGATA mice received i.n. Alt for 48 h. Flow cytometric analysis of digested pericardial FALCs showing %Ki67high B1a and B1b cells (i). Quantification of the % area of Ki67 staining as assessed by whole-mount immunofluorescence staining of mediastinal FALCs (j). ELISA of total IgM within the pleural lavage (k). Data in i–k combined from two independent experiments, symbols represent individual mice, n=8 or 9 per group, symbols in j represent individual clusters. ns not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (normally distributed data analysed by one-way ANOVA with Sidak multiple comparisons post test, non-normally distributed data analysed using the Kruksal–Wallis test with Dunn’s multiple comparisons post test). Error bars represent mean with s.e.m. in all graphs.

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