Fig 1.
A. 1012 fluorescence labelled microspheres were orally administered and after 4h cell subsets in the mLN and the spleen of control and mLN resected mice were analysed. Means and standard error are given from 3–4 independent experiments (significant differences in the paired t-test are indicated by *: P < 0.05). B. Uptake of microspheres in mLN and the spleen in sham-operated or mLN resected mice. Three to four independent experiments were performed. Significant differences in the paired t-test are indicated by *P< 0.05.C. Gating strategy of bead carrying CD19+ cells showed for the spleen.
Fig 2.
Oral application of OVA-CT induces the proliferation of splenic memory IgM+ and marginal zone B cells.
Control and mLN resected mice (n = 3–4) were orally challenged with OVA-CT. 25 days after the challenge the spleen was removed and analysed. A. Representative immunohistological staining of cryosections showing incorporated BrdU (brown) and B cells (blue) in the spleen of control mice +/- OVA-CT administration. Focusing on germinal centers after OVA-CT challenge, antigen specific B cells were stained using Alexa Fluor 555 conjugated OVA (red), IgM (blue) and B220 (green). B. B1 and B2 cells were distinguished by their IgM, CD19, CD5 and CD11b expression. In addition, B2 cells were separated into follicular B cells (IgM+ IgD+ CD21/35+ CD23+), marginal zone B cells (IgM+ IgD- CD21/35++ CD23-) and memory B cells and marginal zone B cells (IgM++ IgD-). Significant differences in the unpaired t-test are indicated by *P< 0.05; ***P< 0.001.
Fig 3.
Splenic IgM+ B cells proliferate after orally applied antigen challenge.
A. Splenic IgM+ B cells of C57BL/6-Ly5.1 mice were isolated using the MACS technique and cytospins of isolated cells were performed. The illustration shows the IgM+ B cells in blue and the proliferating IgM+ B cells (BrdU+ cells) in brown. The purity of the isolated cells was near 100%. B. Flow cytometry analysis of isolated cells. Majority of isolated cells were IgM+ B cells. IgA+ B cells were not detected (n.d). Means and standard error of the mean are given for seven independent experiments.
Fig 4.
Splenic IgM+ B cells migrate into different lymphoid tissues.
Isolated OVA-CT treated IgM+ C57BL/6-Ly.5.1 cells were injected into non-treated (-OVA) and treated (+OVA) mice. Migration of these cells into the gut, PPs, mLN and spleen was analysed by flow cytometry. A. Gating strategy of C57BL/6-Ly5.1 injected cells showed for the spleen, mLN, PPs and gut. B. Inoculated IgM+ B cells migrated into all analysed tissues. Means and standard error are given from 4–5 independent experiments (significant differences in the unpaired t-test are indicated by *: P < 0.05; **: P < 0.01). C. The number of HEL specific B cells was analysed after the cell transfer into WT recipients. Splenic IgM+ B cells were isolated from the MD4 mice, in vitro HEL stimulated and injected into non-treated (-HEL) and treated (+HEL) mice. Means and standard error are given from 4–8 independent experiments (significant differences in the unpaired t-test are indicated by *: P < 0.05; ***: P < 0.001).
Fig 5.
IgM+ B cells that migrate into the gut are OVA-specific IgA+ plasma cells.
The mLN resection increases IgA expression in the spleen, but not in the gut. Isolated OVA-CT treated IgM+ C57BL/6-Ly.5.1 cells were analysed for their immunoglobulin expression by flow cytometry. A. Comparison of isolated cells before and after the transfer into recipients. After migration of the injected cells into the spleen and the gut percentages were compared to the initial cell population. B. IgM+ B cells were injected into non-treated (-OVA) and treated (+OVA) mice and the percentage of different markers expressed on these cells after migrating in the spleen and the gut were analysed using flow cytometry. Significant differences in the unpaired t-test are indicated by *P< 0.05; **P< 0.01; ***P< 0.001. C. The serum of non-treated (-OVA) and treated (+OVA) animals, which received splenic IgM+ B cells, was tested in ELISA for OVA-specific antibodies. OVA-specific antibodies of non-treated animals are equalized as 100%. The measured OVA-specific antibodies of treated animals are calculated as a % change to non-treated (-OVA) mice (x-axis). Means and standard error are given from 3–4 independent experiments (significant differences in the unpaired t-test are indicated by *: P < 0.05). D. IgM+ B cells of OVA/CT treated mice were isolated from the spleen or the mLN and co-transferred to WT mice. After OVA treatment intestines of these mice were analysed and IgA+ B cells from the mLN and spleen were measured. Two independent experiments were performed (n = 4). Significant differences in the paired t-test are indicated by **P< 0.01. E. Recipients underwent the mLN resection. After splenic IgM+ B cell transfer recipients were orally challenged with OVA. Four independent experiments were performed. Significant differences in the unpaired t-test are indicated by *P< 0.05; **P< 0.01; ***P< 0.001.
Fig 6.
Oral administration of HEL activates transferred IgM+ B cells in the gut.
HEL specific IgM+ B cells from MD4 mice were isolated and stimulated in vitro for 24h with HEL. Isolated cells were injected into WT mice. WT mice were subsequently challenged with HEL. Expression of CD80 and CCR9 increased after oral HEL treatment in the gut. Significant differences in the unpaired t-test are indicated by **P< 0.01; ***P< 0.001.