Fig 2: Parasite-specific IgM produced in FALCs is dependent upon IL-33R.(a) Representative immunofluorescence staining of mediastinal FALCs from naive and day 11 Ls-infected BALB/c and Il1rl1-/- mice showing IgM (Green) Ki67 (Red) and B220 (blue). Scale bar, 50 µm. (b,c) Analysis of digested pericardial FALCs from naive and day 11 Ls-infected BALB/c and Il1rl1-/- mice showing total cell number and the number of B1a, B1b and B2 cells (gated as shown in Supplementary Fig. 1). (d,e) Flow cytometric analysis of digested pericardial FALCs from naive and day 11 Ls-infected BALB/c and Il1rl1-/- mice showing frequency of SSChighIgD- B2 plasma cells (d) and quantification of frequency and number of SSChighIgD- B2 plasma cells (e). (f) ELISA of total and Ls antigen-specific IgM in the pleural lavage of naive and day 11 Ls-infected BALB/c and Il1rl1-/- mice. Data are combined from two independent experiments; symbols represent individual mice, n=8 per time point. 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.

Fig 3: FALC activation during acute airway inflammation is IL-33 dependent.(a) ELISA of IL-33 in the pleural lavage (pl Lav) of naive and Alt-treated C57BL/6 mice. Data are combined from two independent experiments, symbols represent individual mice, n=6 or 9 per group. (b) Representative whole-mount immunofluorescence staining of mediastinal FALCs from naive and Alt-treated BALB/c and Il1rl1-/- mice showing IgM (green), Ki67 (red) and B220 (blue) and quantification of the percentage area of Ki67 staining within individual clusters, scale bar, 50 µm. (c,d) Flow cytometric analysis showing proliferating Ki67high and Ki67int (c, left panel) and quantification of % Ki67high (c, right panel) and number (d) of B1a (first row), B1b (second row) and B2 (third row) cells from digested pericardial FALC cells and PLEC from naive and Alt-treated BALB/c and Il1rl1-/- mice. (e) ELISA of total IgM in the pleural lavage (pl Lav), peritoneal lavage (p Lav) and serum of naive and Alt-treated BALB/c and Il1rl1-/- mice. Data in c–e are combined from two independent experiments, symbols represent individual mice, n=8 or 10 per time point. (f) ELISA of total IgM in the supernatant of overnight culture of PLEC and digested FALC cells from naive and Alt-treated BALB/c mice. Data are combined from three independent experiments, symbols represent individual mice, n= 9 or 14 per time point. 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.

Fig 4: FALC B cells secrete parasite-specific IgM locally upon infection.(a,b) Peritoneal lavage (p Lav), pleural lavage (pl Lav) and serum of naive and day 11 Ls-infected C57BL/6 mice were assessed for the presence of Ls antigen specific (a) and total IgM (b) by ELISA. Data shown combined from two representative experiments of >5 independent experiments n=6 or 12 per group. (c) Location of the pericardium and mediastinum within the pleural cavity, thymus (T), Lungs (L) and Heart (H). (d) Representative whole-mount immunofluorescence staining of mediastina of naive and day 11 Ls-infected C57BL/6 mice showing CD45+ clusters in green imaged with a fluorescence stereomicroscope. Original magnification × 4. (e) Quantification of the number of FALCs per mediastinum (left) and mesenterium (right) of naive, day 8 and day 11 Ls-infected C57BL/6 mice. Data combined from at least two independent experiments; symbols represent individual mice, n=11 per time point. (f) Flow-cytometric analysis of digested pericardial FALC cells and PLEC of naive and day 11 or 18 Ls-infected C57BL/6 mice. Numbers of each B-cell subset were quantified. Data combined from two independent experiments, symbols represent individual mice, n=12 per time point. (g) Isolated peritoneal exudate cell (PEC), PLEC, FALC and lymph node (LN) cells of naive and day 18 Ls-infected C57BL/6 mice were cultured overnight and Ls antigen-specific IgM production quantified by ELISA. Data representative of two independent experiments; symbols represent individual mice n=2, 4, 5 or 6 per group. (h) B1a, B1b and B2-cell populations isolated from pericardial and mediastinal FALCS from five individual day 18 Ls-infected C57BL/6 mice were cell-sorted as in (Supplementary Fig. 1) and total and Ls-specific IgM secretion was determined for each population by ELISA. The relative quantity of total and Ls-specific IgM produced by each cell type is shown. Data combined from two independent experiments. 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.

Fig 5: FALC B2 cells differentiate into plasma cells in parasite infected mice.(a,b) Representative immunofluorescence staining of mediastinal FALCs of naive and day 11 Ls-infected C57BL/6 mice showing Ki67 (red), B220 (blue) and IgM (green in a) or CD138 (green in b) and quantification of the percentage area of Ki67 staining (a). Scale bar, 50 µm. (c–e) Flow-cytometric analysis of digested pericardial FALC cells and PLEC from naive and day 11 Ls-infected C57BL/6 mice showing Ki67high B2 cells (c, upper panel); and SSChighIgD- plasma cells (c, lower panel). Quantification of % of Ki67high and Ki67int (d) and plasma cells (e, left graph) in B2 cells and plasma cell number (e, right graph) in pericardial FALCs, PLEC and lymph node (LN) are shown. (f) Flow-cytometric analysis of digested pericardial FALC cells from naive and day 11 Ls-infected C57BL/6 mice showing Ki67highGL-7+ germinal centre (GC) B cells and quantification of germinal centre B cells as a % of the total B2-cell population. Data in a–f are combined from two independent experiments, symbols in the graph in a represent individual clusters, data in d–f represent individual mice, n=12 per time point. ns not significant, **P<0.01, ****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.