TACI, BCMA and BAFF-R expression by mucosal B-1 (CD5⁺ B220⁺) B cells.

<p>Wild-type mice were nasally immunized with TNP-LPS with or without nCT, nCT alone or PBS. (A) Five days after immunization, mononuclear cells were isolated and stained for CD5, B220, TACI, BCMA and BAFF using the respective fluorescence-conjugated mAbs as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025396#s2" target="_blank">Materials and Methods</a> section. Samples were then subjected to flow cytometric analysis by FACSCalibur®. *<i>p</i><0.05 when compared with mice given TNP-LPS or nCT alone. The values are presented as the mean ± SEM of 10 mice for each group and represent a total of five separate experiments. (B) B-1 B cells were purified by FACSAria from SMGs, NPs and NALT of mice given nasal TNP-LPS with or without nCT two days after the immunization. The expression of TACI, BCMA and BAFF-R mRNA by B1-B cells was assessed by quantitative real-time PCR. Relative expression of TACI, BCMA and BAFF-R mRNA are displayed as the fold change of respective transcript expression by the experimental group (TNP-LPS plus nCT) over the expression by controls (TNP-LPS alone). The values are presented as the mean ± SEM of 10 mice for each group and represent a total of five separate experiments.</p

APRIL and BAFF expression by CD11c⁺ DCs.

<p>Mice were nasally immunized with TNP-LPS with or without nCT, nCT alone or PBS. (A) Five days after immunization, mononuclear cells were isolated from SMGs, NPs and NALT and stained for CD11c, APRIL and BAFF using the respective fluorescence-conjugated mAbs described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025396#s2" target="_blank">Materials and Methods</a> section. Samples were subjected to flow cytometric analysis by FACSCalibur®. *<i>p</i><0.05 when compared with mice given TNP-LPS or nCT alone. The values are presented as the mean ± SEM of 10 mice for each group and represent a total of five separate experiments. (B) CD11c⁺ DCs were isolated from SMGs, NPs and NALT of mice given nasal TNP-LPS with or without nCT by AutoMACS two days after the immunization. The expression of BAFF- and APRIL-specific mRNA by mucosal DCs was determined by quantitative real-time PCR. Relative expression of BAFF- and APRIL- specific mRNA were displayed as the fold change of respective transcript expression by experimental group (TNP-LPS plus nCT) over the expression by controls (TNP-LPS alone). The values are presented as the mean ± SEM of 10 mice for each group and represent a total of five separate experiments.</p

The frequencies of CD11c⁺ DCs and co-stimulatory molecule expression in wild-type mice given nasal TNP-LPS with or without nCT^a.

a<p>Mononuclear cells from SMGs, NPs and NALT were isolated five days after immunization with TNP-LPS plus nCT or TNP-LPS alone, and were stained with FITC-conjugated anti-CD11c mAb.</p>b<p>Mononuclear cells were stained with FITC-conjugated anti-CD11c and PE-labeled anti-CD40, -CD80, -CD-86 or -MHC class II mAbs and were then subjected to flow cytometry analysis by FACSCalibur®.</p><p>*<i>p</i><0.05 compared with immunized mice given TNP-LPS alone.</p

Expression of AID, αCT, Iμ-Cα transcripts in B-1 B cells from the oral-nasopharyngeal effector and inductive tissues of mice nasally immunized with TNP-LPS with/without nCT or nCT alone.^a^,^b^,^c

a<p>Five days after nasal immunization, B-1 B (CD5⁺ B220⁺) cells from the SMGs, NPs and NALT (as a positive control) were purified by FACS and were then subjected to semi-quantitative RT-PCR.</p>b<p>The numbers are mean the expression percentages of the respective CSR-associated molecules when the density of β-actin expression on respective lymphoid tissues was calculated as 100 with ChemiDoc XRS Quantity one Analysis software (Bio-Rad).</p>c<p>The values are presented as the mean ± SEM of 10 mice for each group and represent a total of five separate experiments.</p><p>*<i>p</i><0.05 when compared with mice given TNP-LPS alone.</p>#<p><i>p</i><0.05 when compared with mice given nCT alone.</p

Induction of IgA CSR by B-1 B cells in the presence of mucosal DCs.

<p>IgA negative B cells were purified from the peritoneal cavity of wild-type mice by magnetic sorting. Purified cells (8×10⁶ cells/ml) were then cultured with DCs (4×10⁵ cells/ml) from SMGs, NPs and CLNs of mice given nasal TNP-LPS with/without nCT or nCT alone in the presence of 1 ng/ml TGF-β₁, and 100 ng/ml IL-5. After 5 days of incubation, non-adherent cells were harvested and stained with FITC-conjugated anti-IgA, and PE-labeled anti-CD5 mAbs. The data represent a typical profile of 10 mice for each group.</p

Potential Roles of CCR5⁺ CCR6⁺ Dendritic Cells Induced by Nasal Ovalbumin plus Flt3 Ligand Expressing Adenovirus for Mucosal IgA Responses

<div><p>We assessed the role of CCR5⁺/CCR6⁺/CD11b⁺/CD11c⁺ dendritic cells (DCs) for induction of ovalbumin (OVA)-specific antibody (Ab) responses following mucosal immunization. Mice given nasal OVA plus an adenovirus expressing Flt3 ligand (Ad-FL) showed early expansion of CCR5⁺/CCR6⁺/CD11b⁺/CD11c⁺ DCs in nasopharyngeal-associated lymphoid tissue (NALT) and cervical lymph nodes (CLNs). Subsequently, this DC subset became resident in submandibular glands (SMGs) and nasal passages (NPs) in response to high levels of CCR-ligands produced in these tissues. CD11b⁺/CD11c⁺ DCs were markedly decreased in both CCR5^−/− and CCR6^−/− mice. Chimera mice reconstituted with bone marrow cells from CD11c-diphtheria toxin receptor (CD11c-DTR) and CCR5^−/− or CD11c-DTR and CCR6^−/− mice given nasal OVA plus Ad-FL had elevated plasma IgG, but reduced IgA as well as low anti-OVA secretory IgA (SIgA )Ab responses in saliva and nasal washes. These results suggest that CCR5⁺CCR6⁺ DCs play an important role in the induction of Ag-specific SIgA Ab responses.</p> </div

OVA-specific Ab responses in systemic and mucosa-associated lymphoid tissues of chimera mice which lack CCR5- or CCR6-expressing DCs (Figure S1).

<p>(A and C) CD11c-DTR, CD11c-DTR/C57BL/6, CD11c-DTR/CCR5^−/− and CD11c-DTR/CCR6^−/− mice were injected with DT via the intraperitoneal route 6 h before each nasal immunization (three times at weekly intervals with OVA plus Ad-FL). CD11c-DTR/C57BL/6 chimera mice given the DT injection served as positive controls. (B) CD11c-DTR/C57BL/6, CD11c-DTR/CCR5^−/−, CD11c-DTR/CCR6^−/− and normal C57BL/6 were nasally immunized three times at weekly intervals with OVA plus Ad-FL without DT injection. (A and B) Seven days after the last immunization, levels of SIgA anti-OVA Abs in saliva, SIgA and IgG Abs in NWs, and IgA, IgG and IgM Abs in plasma were determined by OVA-specific ELISA. (C) MNCs from NPs, SMGs and spleens were isolated 7 days after the last immunization and subjected to OVA-specific ELISPOT assay to determine the numbers of IgA, IgG and IgM AFCs. The values shown are the mean ± SEM taken from five separate experiments with a total of 25 mice in each experimental group. The data for controls were obtained from two separate experiments which consisted of 6 mice for each experiment. The ELISA and ELISPOT data for spleen represented the Ab responses from 12 individual mice for controls and 25 individual mice for experimental groups. MNCs from SMGs and NPs were pooled from 2 or 3 mice and subjected to OVA-specific ELISPOT assays. N.D. means that O.D. values were not detected. (A and C) *<i>p</i><0.05 when compared with DT treated, CD11c-DTR/C57BL/6 chimera mice nasally immunized with OVA plus Ad-FL. (B) No significant differences were detected when compared with normal C57BL/6 mice.</p

Kinetics of chemokine receptor expression by CD11b⁺ DCs.

<p>Mice were nasally immunized weekly for three consecutive weeks with OVA plus Ad-FL (A-D). Five, 10, 14 and 21 days after the initial immunization, mononuclear cells (MNCs) from NALT (A), CLNs (B), NPs (C), and SMGs (D) were stained with FITC-conjugated anti-CD11b, PE-labeled anti-CCR5, allophycocyanin-tagged anti-CCR6 or -CCR7 and biotinylated anti-CD11c mAbs followed by PerCP-Cy5.5-streptavidin. CD11c⁺ and CD11b⁺ cells were gated and their CCR expression was analyzed by FACSCalibur®. The values shown are the mean of the actual numbers of cells ± SEM taken from five separate experiments (two samples/experiment) with a total of 10 samples in each experimental group. * <i>p</i><0.05, ** <i>p</i><0.01 when compared with day 0.</p

CCL3, CCL4, CCL5 and CCL20 synthesis in various mucosal tissues.

<p>Mice were nasally immunized weekly for either two or three consecutive weeks with OVA plus Ad-FL. Three days after the second immunization (day 10) or one week after the last immunization (day 21), MNCs were taken from NALT, NPs and SMGs, and cultured <i>in vitro</i> for 5 days. Culture supernatants were harvested after 5 days of incubation and analyzed for the respective chemokine by ELISA. The values shown are the mean ± SEM taken from five separate experiments with a total of 25 mice in each experimental group. * <i>p</i><0.05, ** <i>p</i><0.01 when compared with naïve mice (day 0).</p

Comparison of the frequency of CD11b⁺ DCs in mucosal and peripheral lymphoid tissues of normal, CCR5^−/− and CCR6^−/− C57BL/6 mice.

<p>Mice were nasally immunized weekly for three consecutive weeks with OVA plus Ad-FL. (A) One week after the last immunization or (B) three days after the second immunization (day 10), MNCs were taken from NALT, CLNs, NPs and SMGs, and stained with FITC-conjugated anti-CD11b and PE-labeled anti-CD11c. All samples were subjected to flow cytometry analysis with a FACSCalibur®. After lymphocyte gating, (A) 10,000 cells were assessed whereas (B) 50,000 cells were taken for the analyses since the frequencies of CD11b⁺ DCs at day 10 samples were limited. The results represent the mean values ± SEM from three separate experiments with a total of 15 mice in each experimental group and are taken from three separate experiments. The profiles represent typical results and are taken from one of three separate experiments. *<i>p</i><0.05 when compared with normal C57BL/6 mice.</p