Fig 2: Splenic IgE+ cells are responsible for IgE production in Spib-/- mice. (A) Gating strategy of CD3- CD4- CD45+ live lymphocytes. (B) The numbers of CD45+ live lymphocytes of Peyer's patches (PPs), the mesenteric lymph node (MLN), and the spleen from OVA-sensitized mice (day 20). (C,D) The CD3- CD4- 7-AAD- CD45+ cells were further divided into IgE+ B220- and IgE+ B220- cells. The bar graphs show the number of IgE+ B220- and IgE+ B220- cells in the MLN (C) and the spleen (D). Representative data from at least three independent experiments are shown. Data are expressed as mean ± SEM (n = 5 or 6). *p < 0.05, **p < 0.01, calculated by unpaired t-test for B and Kruskal–Wallis test for C,D.

Fig 3: (A) Toluidine blue staining of mast cells. The arrows indicate mast cells. (B) Bar graph represent the number of mast cells counted per 350 µm2 at a magnification of x 400 in the epidermis region were counted (C) Serum IgE. (Scale bar = 100 µm; magnification: x 100) (i- Control, ii- Compound 1 alone, iii- Compound 2 alone, iv- DNCB, v- DNCB+ Compound 1, vi- DNCB+ Compound 2) Data represent the mean ± SEM (n = 3). ***p < 0.001 vs. control; #p < 0.5, ##p < 0.01 vs. DNCB.

Fig 4: Enhanced recruitment and maturation of B cells in KPC compared to orthotopic tumors. (A) Density (number of cells per gram tissue) of CD45+ CD19+ B cells quantified by flow cytometry in KPC tumors (n = 10) and orthotopic tumors (n = 19). (B) Proportion of CD19+ B cells out of CD45+ cells quantified by flow cytometry in KPC tumors (n = 10) and orthotopic tumors (n = 19). (C) Proportion of B cells (CD19+/B220+) and T cells (CD3+) out of CD45+ cells in KPC mice. Black circles: untreated KPC mice; Half circles: PBS-treated KPC mice at endpoint; white circles: PBS-treated KPC mice not at endpoint. The association was assessed using a Pearson correlation. (D) Flow cytometry gating strategy for activated B cells by GL7hi expression on CD45+ B220+ B cells, with fluorescence minus one (FMO) control used to assess background. (E,F) Flow cytometry quantification of proportion of activated B cells by GL7hi cells out of total B220+ B cells in (E) the healthy pancreas (n = 6) and KPC tumor (n = 7) and (F) in the MLN and spleen or healthy (n = 3) and KPC (n = 6) mice. Mean + standard deviation (SD), Unpaired t-test. (G) Flow cytometry quantification of the percentage of CD95+ cells out of GL7hi CD19+ cells in KPC tumors (n = 4). (H) Flow cytometry quantification of B cell subsets out of total B cells isolated from KPC tumors: B1 cells (CD11b+ B220+) (n = 3), memory IgG1 (n = 6), memory IgG2a/b (n = 6), memory IgG3 (n = 3), memory IgA (n = 3), memory IgE (n = 3), IL-10+ B cells, post ex vivo stimulation with LPS, and PMA (n = 9). (I) Flow cytometry gating strategy for plasmablasts CD138lo and plasma cells by CD138hi expression on CD45+ cells, with FMO control used to assess background. (J) Flow cytometry quantification of proportion of CD138hi plasma cells out of total CD45+ immune cells in the spleen of healthy (n = 10) and KPC (n = 11) mice. (K,L) Concentration of immunoglobulin isotypes analyzed by Mesoscale (IgA - IgM) and ELISA (IgE) present in (K) the plasma and (L) pancreas tissue lysates of healthy (n = 5) (white) and KPC (n = 5) mice (black). (M) Relative concentration of C1q-Ig immune complexes as measured by ELISA. Negative and positive controls were provided by the ELISA kit manufacturer, dashed line represents threshold for positivity. The concentration of immune complexes was measured in healthy control pancreata (n = 5) and KPC tumors (n = 4). A confirmation test that disrupts ICs was performed for each sample as recommended by the manufacturer, however, results show no difference since no ICs were detected. Each data point represents an individual mouse sample, mean and SD are also indicated. Statistical significance was analyzed by unpaired t-test where *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig 5: Plpp6-/- mice have reduced lung inflammatory responses to allergen(A–D) Plpp6-/- and WT mice were airway sensitized and challenged with HDM and their immunophenotype was determined on protocol day 16—a time of increased allergic lung inflammation. (A) Representative images of lung histology after staining with hematoxylin and eosin (H&E), arrowheads indicate leukocyte infiltration (left panels), or with periodic acid schiff (PAS), asterisks indicate mucous cells metaplasia (right panels); scale bars correspond to 100µm in 10x images (H&E) and 20x images (PAS) and 50µm in 40x images. (B) Lung differential cell numbers of eosinophils (Eos) and neutrophils (PMN) were determined by FACS (representative contour plots on the left; numbers in red represent the percentage of leukocytes); experiments were performed 3 times for a total of n = 9. (C) Lung differential cell numbers of alveolar macrophages (aMacs) and T cells determined by FACS (representative contour plots on the left; numbers in red represent the percentage of leukocytes); experiments were performed 3 times for a total of n = 9 (see Figure S3A for gating strategy). (D) Gene expression of il4, il5, and il13 by RT-qPCR in lung tissue harvested on protocol day 16; data represent fold change in gene expression with HDM relative to naive within the same genotype; experiments were performed 3 times for a total of n = 4 to 5.(E) Total serum IgE was measured by ELISA on days 4 and 16 of the HDM protocol; experiments were performed 2 times for a total of n = 3 to 5.(F) Resistance of the respiratory system (Rrs) was determined by invasive measurement in anesthetized animals by Flexivent in response to increasing doses of methacholine (MCh) (1, 3, 10, 30, and 100 mg/mL); experiments were performed 2 times for a total of n = 4 to 9; data represent percent change from baseline. *p < 0.05, **p < 0.01 and*** p < 0.001 comparing WT and Plpp6-/-, and #p < 0.05 comparing HDM and naive within same mouse genotype by unpaired nonparametric Mann-Whitney test. Bars represent median with interquartile range in (B–E) and mean ± SE in (F). See also Figure S3.