(A) Flow cytometry of BM, spleen (SPL), blood, and PerC cells incubated with fluorescent (FITC), 1 μm beads and then stained with anti-B220 and anti-CD19. Dot plots show the gated population of CD19⁺ cells. Phag⁺, Phagocytic. Data are representative of four independent experiments (n=6). (B) Percentage of phagocytic B cells in BM, spleen, blood, and PerC. Cells were incubated with fluorescent, 1 μm beads or pHrodo E. coli bioparticles and then stained with anti-B220, anti-CD19, anti-IgD, anti-IgM, and anti-CD11b (PerC) or anti-CD23 (BM, spleen, and blood). Results are expressed as mean ± sem of six mice from two independent experiments.

(A) Flow cytometry of PerC cells incubated in vitro with fluorescent, 1 μm beads and then stained with anti-B220, anti-CD19, anti-CD11b, anti-CD5, and anti-IgD antibodies. Percentages of phagocytic cells are shown in red. (B) Percentage of phagocytic cells in PerC B cells incubated in vitro with fluorescent, 1 μm beads or pHrodo E. coli bioparticles, stained with the aforementioned antibodies (A), and analyzed by flow cytometry. Results are mean ± sem of 10 mice from four independent experiments. (C) In vivo analysis of phagocytosis by PerC B cells. Mice were injected i.p. with 1 μm beads or pHrodo E. coli bioparticles. After 3 h, PerC cells were collected, stained with the aforementioned antibodies (A), and analyzed by flow cytometry. Results are presented as percentage of phagocytic cells and are mean ± sem from three independent experiments (n=5). (B and C) Statistical differences were determined by unpaired Student's t test. *P < 0.05; **P < 0.01; ***P < 0.001. (D) PerC cells were incubated with fluorescent, 1 μm beads in the presence of various concentrations of cytochalasin D and stained with the aforementioned antibodies (A). Results are presented as the percentage of phagocytic cells relative to that of PBS-treated controls. Results are expressed as mean ± sem of four independent experiments (pool of cells from three mice/experiment). (E) Representative IFC images of PerC B-1 B cells incubated with fluorescent, 1 μm beads, stained with anti-IgM and anti-CD5, and acquired using an ImageStream multispectral flow cytometer. Black arrows point to internalized beads. BF, Bright field. (F) Percentage of internalized versus surface-bound beads in FITC-associated B cell subsets. Data were generated using Ideas software. Results are expressed as mean ± sem of five independent experiments (pool of cells from five mice/experiment).

TEM of PerC B-1 B cells incubated with fluorescent, 1 μm beads (red ″X″). Phagocytic B-1a (upper) and B-1b (lower) cells were sorted, fixed, processed, and analyzed by TEM. Original scale bar, 2 μm. Original magnification, 12,000×. Data are representative of two independent experiments (pool of cells sorted from six mice/experiment).

(A) DIC images (upper) and fluorescence microscopy (lower) of sorted B-1a and B-1b cells incubated with nonfluorescent, 1 μm beads, fixed and stained with anti-mouse LAMP1 (green). Nuclei were stained with DAPI (blue). Left rows are cells without ingested beads, whereas center and right rows show cells with ingested beads. LAMP1 is seen colocalizing with ingested beads (black arrows in DIC images point to internalized beads). Original scale bar, 2 μm. Original magnification, 1000×. (B) IFC images of PerC B-1a and B-1b cells loaded with fluorescent dextran (green) and incubated with nonfluorescent 1 μm or 2 μm beads. Cells were incubated with DAPI for visualization of nuclei (blue). Upper rows are cells without ingested beads, whereas middle and bottom rows show cells with ingested 1 μm and 2 μm beads, respectively. Dextran is seen colocalizing with ingested beads (black arrows in bright field images point to internalized beads). (A and B) Representative of three independent experiments (pool of cells from six mice/experiment). (C) Intracellular bacterial killing by sorted PerC B cell subsets incubated with live E. coli; attached bacteria were killed by incubation with gentamicin, after which, leukocytes were lysed at different time-points (time, horizontal axis). Live bacteria in lysates were counted after inoculation on LB agar plates. Results are expressed as mean ± sem of four independent experiments (pool of cells from at least 10 mice/experiment).

(A) Representative histograms of CD4⁺ T cell proliferation (percentage of CFSE dilution) after 3 days in coculture with sorted PerC B cell subsets or SPL preincubated for 12 h with 1 μm OVA-beads (1 μg OVA/well). Percentage of proliferative CD4⁺ T cells is shown in each histogram. “NONE” panel represents T cells incubated with 1 μm OVA-beads without APCs. (B and C) Sorted PerC B cells and SPL were incubated for 12 h with 1 μm (left) or 6 μm (right) OVA-beads (1 μg OVA/well), plain beads, or OVA-beads SN and then cocultured with CFSE-labeled OT-II CD4⁺ T cells for 3 days at a 1:2 ratio. Percentage of divided CD4⁺ T cells (B) and IL-2 secreted by CD4⁺ T cells (C) was measured by FACS and ELISA, respectively. (D and E) Sorted PerC B cells and SPL were incubated with 1 μm (left) or 6 μm (right) OVA-beads in the presence of chloroquine or anti-mouse I-A/I-E (MHC II block) prior to coculture with OT-II CD4⁺ T cells for 3 days. Percentage of divided CD4⁺ T cells (D) and IL-2 secreted by CD4⁺ T cells (E) was measured (as described for B and C, respectively). Results are expressed as mean ± sem of at least four independent experiments (pool of cells from 10 mice/experiment).

Sorted PerC B cells, PerC macrophages, or BMDCs were incubated for 12 h with different amounts of 1 μm OVA-beads or soluble OVA and cocultured with OT-II CFSE-CD4⁺ T cells for 3 days at a 1:1 ratio. Percentage of divided CD4⁺ T cells (A) and IL-2 secreted by CD4⁺ T cells (B) was measured by FACS and ELISA, respectively. Data are from one representative experiment of three performed.