Vitamin B9 carrier-mediated pathway is not specific pathway in the maintenance of T cell survival.

<p>CD25⁺ CD4⁺ T cells were cultured with an anti-CD3 antibody in complete medium containing 100 nM methotrexate (MTX), and the frequency and absolute cell numbers of Foxp3⁺ and Foxp3⁻ CD4⁺ T cells were determined. Data are means ± SEM (n = 4). Data are representative of two independent experiments.</p

Vitamin B9 is IL-2-independent survival factor for Treg cells.

<p>(A) The amounts of intracellular vitamin B9 were measured using purified CD4⁺FR4^hi Treg or CD4⁺FR4^low/− non-Treg cells. Data are means ± SEM (n = 4). (B, C) Experiments similar to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032094#pone-0032094-g001" target="_blank">Fig. 1B</a> were performed in the presence of anti-CD3 antibody stimulation with or without IL-2 stimulation. Cell number of Foxp3⁺CD4⁺ T cells (B) and the expression of phosphorylated STAT5 (pSTAT5) in Foxp3⁺CD4⁺ T cells (C) were determined. Data in (B) are means ± SEM (n = 6). Similar results were obtained from 3 separate experiments.</p

Requirement of vitamin B9 for the maintenance of Treg cells.

<p>(A) Purified naïve CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 antibodies plus TGF-β in the presence of normal [Vit B9(+)] or reduced [Vit B9(−)] amounts of vitamin B9. After 4 days, total cell numbers were calculated, and the differentiation into Foxp3⁺ Treg cells was examined by flow cytometry. Data are means ± SEM (n = 4). (B) CD25⁺CD4⁺ T cells were cultured with anti-CD3 antibodies in Cont or B9(−) medium. The frequencies of Foxp3⁺ and Foxp3⁻CD4⁺ T cells (B) were determined by flow cytometry. Cell numbers were calculated using the total cell number and flow cytometric data. Data are means ± SEM (n = 6). (C) Experiments similar to that shown in (B) were performed with different concentrations of vitamin B9. The relative cell number of Foxp3⁺ Treg cells is expressed as a ratio to the cell number in control medium. The values and means are indicated with dots and lines, respectively. Similar results were obtained from 2 independent experiments.</p

Depletion of dietary vitamin B9 selectively reduces Treg cells in the small intestine.

<p>Mice were maintained on a control [Vit B9(+)] or vitamin B9-depleted [Vit B9(−)] diet for 8 wk. (A) Vitamin B9 concentrations were measured in intestinal washes of the small intestine (SI), large intestine (LI), and serum. The data are mean ± SEM (n = 6). (B, C) The frequency and cell numbers of Foxp3⁺ and Foxp3⁻ CD4⁺ T cells in the small intestine (B), colon, and spleen (C) were calculated using the total cell number and flow cytometric data (mean ± SEM, n = 6). (D) Flow cytometric analysis was performed to determine the expression levels of Foxp3, CTLA4, and GITR on the surface of FR4^low/− (thin line) and FR4^hi (thick line) CD4⁺ T cells in the LP. Similar results were obtained from 3 separate experiments.</p

MOESM1 of Intranasal administration of cationic liposomes enhanced granulocyte–macrophage colony-stimulating factor expression and this expression is dispensable for mucosal adjuvant activity

Additional file 1: Figure S1. DOTAP/DC-chol liposomes potentiate both mucosal and systemic OVA-specific antibody responses. The data show the OVA-specific nasal IgA and serum IgGs for each immunized group (PBS only, OVA alone, or OVA plus liposomes). The data were obtained from three independent experiments. The statistically significant value (*p < 0.0001) shown were calculated from the Kruskal–Wallis test with Dunn’s post hoc test

Role of <i>Lactobacillus pentosus</i> Strain b240 and the Toll-Like Receptor 2 Axis in Peyer's Patch Dendritic Cell-Mediated Immunoglobulin A Enhancement

<div><p>Lactic acid bacteria are well known to possess immune-modulating effects, but the mechanisms underlying their modulation of the gut immune system are not fully understood. Here, we examined the localization of heat-killed <i>Lactobacillus pentosus</i> strain b240 (b240) in intestinal tissues and the effect of b240 on adaptive immune cascades in the gut. Histological analysis showed that b240 co-localized with dendritic cells (DCs) in the subepithelial dome region of Peyer's patches (PPs). In a PP cell culture system, b240 promoted the production of immunoglobulin A (IgA), interleukin (IL)-6, IL-10, interferon (IFN)-γ, and tumor necrosis factor, but not IL-4, IL-5, B-cell activating factors, IFN-α, IFN-β, and transforming growth factor-β1. The enhanced IgA production by b240 was attenuated by neutralizing IL-6, a potent IgA-enhancing cytokine. b240 stimulated DCs to produce an elevated amount of IL-6 in a Toll-like receptor (TLR) 2-, but not TLR4- or TLR9-dependent manner. Finally, we demonstrated that TLR2-mediated IL-6 production from PP DCs in response to b240 activated B cells to produce a large amount of IgA in a DC-B cell co-culture system. Our findings open up the possibility that the heat-killed form of <i>Lactobacillus pentosus</i> strain b240 can be used as a TLR2-mediated DC-activating biologic for enhancing IgA production in the intestine.</p></div

IL-6 producing cells in the PPs in response to b240.

<p>(A) Purified CD11c⁺B220⁻ cells as DCs, CD4⁺ cells as T cells, CD19⁺ cells as B cells from the PPs, or PP cells (1×10⁵ cells) were cultured with (black) or without (white) heat-killed b240 (1.6×10⁶ counts) for 3 days. IL-6 in the culture supernatants was determined by CBA. Data are expressed as mean ± SEM (n = 3). ND, not detected. The detection limit was 20 pg/ml. ^*<i>P</i><0.05 by Student's <i>t</i>-test. Data are representative of 2 independent experiments producing similar results. (B) After the ligated intestinal loop assay with 1 mg/ml of FITC-labeled b240, histological analysis was performed to examine the co-localization of b240 (green) with DCs in PP. Tissue sections were prepared and stained with anti-CD11c antibody (red) and 4,6-diamidino-2-phenylindole (blue). Magnification of the area in the first image (white square) shows the contact between b240 and CD11c⁺ cells (arrowhead). White and yellow bars indicate 100 and 25 μm, respectively. (C) After PP CD11c⁺B220⁻ DCs were cultured with FITC-labeled b240 on a Cell Desk, the interaction between PP CD11c⁺B220⁻ DCs and b240 was analyzed using a fluorescence microscope. The bar indicates 10 μm.</p

Important factor for the enhancement IgA production from PP cells by b240.

<p>(A) PP cells (1.5×10⁶ cells) were cultured with saline (open circles), 1.2×10⁶ counts of heat-killed b240 (closed squares), or 1.2 × 10⁷ counts of heat-killed b240 (closed circles) for 1, 3, 5, and 7 days. (B, C) In the presence or absence of heat-killed b240 (4.7×10⁶ counts), PP cells (5.8×10⁵ cells) were cultured with (B) anti-IL-6 mAb (10 μg/ml), anti-IFN-γ mAb (10 μg/ml), anti-TNF mAb (10 μg/ml), rat IgG1 k isotype control (10 μg/ml), (C) LE540 (1 μM), BCMA-Ig+ TACI-Ig (5 μg/ml each), dimethyl sulfoxide, or human IgG1 Fc antibody (10 μg/ml) for 4 days. The stimulation index of each sample was calculated (for example, (b240-treatment and anti-IL-6 Ab treatment)/(saline-treatment and anti-IL-6 Ab treatment) is the stimulation index for anti-IL-6 Ab treatment). (D) PP cells (5.8×10⁵ cells) were cultured with a low dose (light gray), medium dose (dark gray), and high dose (black) of rIL-6 (0.4, 2, or 10 ng/ml), rIFN-γ (0.6, 3, or 15 ng/ml), rTNF (0.08, 0.4, or 2 ng/ml), or heat-killed b240 (4.7×10⁶ counts) for 4 days. IgA or cytokine in the culture supernatants was determined by ELISA or CBA. Data are expressed as mean ± SEM (n = 3). (A, B) ^*<i>P</i><0.05 versus control group by Dunnett's test. (C) Student's <i>t</i>-test was conducted. (D) Statistical analysis was not conducted. Data are representative of 2 independent experiments producing similar results.</p

Induction of PspA-specific systemic and respiratory antibody responses by intranasal immunization with PspA-C-CPE.

<p>Mice were nasally immunized with vehicle, PspA alone, or PspA-C-CPE (PspA; 5 μg) once weekly for 3 weeks. One week after the last immunization, PspA-specific serum IgG (A), nasal IgA (B), BALF IgG (C), and IgA (D) were measured by ELISA. Data are shown as mean ± SEM and are representative of two independent experiments. Vehicle, n = 4; PspA, n = 5; PspA-C-CPE, n = 5. Values were compared by using the non-parametric Mann–Whitney <i>U</i> test. *<i>P</i> < 0.01.</p

Construction and preparation of PspA-C-CPE.

<p>(A) Schematic illustration of PspA-C-CPE. PspA was fused with C-CPE at its N-terminus. A G4S linker was inserted between PspA and C-CPE. (B) PspA and PspA-C-CPE were expressed in <i>Escherichia coli</i> as His-tagged proteins and purified by Ni-affinity chromatography. The PspA and PspA-C-CPE recombinant protein were applied to SDS-PAGE followed by staining with Coomassie brilliant blue. Lane 1, size ladder; lane 2, PspA; lane 3, PspA-C-CPE.</p