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 essential for the survival of Treg cells.

<p>CD25⁺CD4⁺ T cells were cultured with anti-CD3 antibodies in Vit B9(+) or Vit B9(−) medium. The expression of Ki67 (A) and Bcl2 (B) in Foxp3⁺CD4⁺ T cells were determined by flow cytometry (top panels) and graphs show the means fluorescent intensity (MFI; bottom panels). Data are means ± SD (n = 3). Data are representative of 4 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

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

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

Immunohistochemical staining of the OE and OBs after nasal administrationof 30 μg CTB.

<p>OE and OBs were obtained at 0 (untreated), 24, or 72 h after nasal administration of 30 μg CTB. (<b>A</b>) Frozen sections of OE were stained with an anti-CTB Ab (green) or anti-OMP Ab (red). White arrows indicate the olfactory nerve layer. The number of OMP-positive cells (mean ± 1 SD; <i>n</i> = 3 mice) in each experimental group is shown at the right. (<b>B</b>) Frozen sections of OBs were stained with an anti-CTB Ab. (<b>C</b>) Frozen sections of OBs were stained with an anti-OMP Ab (converted to black and white images). Glomeruli at the dorsal and lateral surfaces of the OBs (indicated by the boxes in panel B) are shown. The signal intensity (mean ± 1 SD; <i>n</i> = 3 mice) in each experimental group is shown. Data are representative of three independent experiments. Scale bars: (A, C) 20 μm, (B) 200 μm.</p

Nasal Administration of Cholera Toxin as a Mucosal Adjuvant Damages the Olfactory System in Mice

<div><p>Cholera toxin (CT) induces severe diarrhea in humans but acts as an adjuvant to enhance immune responses to vaccines when administered orally. Nasally administered CT also acts as an adjuvant, but CT and CT derivatives, including the B subunit of CT (CTB), are taken up from the olfactory epithelium and transported to the olfactory bulbs and therefore may be toxic to the central nervous system. To assess the toxicity, we investigated whether nasally administered CT or CT derivatives impair the olfactory system. In mice, nasal administration of CT, but not CTB or a non-toxic CT derivative, reduced the expression of olfactory marker protein (OMP) in the olfactory epithelium and olfactory bulbs and impaired odor responses, as determined with behavioral tests and optical imaging. Thus, nasally administered CT, like orally administered CT, is toxic and damages the olfactory system in mice. However, CTB and a non-toxic CT derivative, do not damage the olfactory system. The optical imaging we used here will be useful for assessing the safety of nasal vaccines and adjuvants during their development for human use and CT can be used as a positive control in this test.</p></div

Nasal NKp46⁺ cells are NK-lineage cells.

<p>a. Flow cytometry of CD45⁺ cells from nasal passage, spleen, lung, and Nasopharynx associated lymphoid tissue (NALT) of <i>Ncr1</i>^<i>GFP/+</i> mice stained with CD3. Numbers in quadrants indicate the percentages of cells in each. Dot plot below shows percentage of GFP (NKp46)⁺ cells in CD45⁺ cells from nasal passage, spleen, lung, and NALT. b. Flow cytometry of CD3⁻NKp46⁺ cells from spleen, lung, and nasal passages stained with CD122, NK1.1, 2B4, CD49b, and CD127. Continuous lines, specific antibodies; Dashed lines, isotype-matched control antibodies. Dot plot below shows the percentage of positively stained cells. Bar, mean; n.s.; not significant (Mann-Whitney <i>U</i> test with Ryan’s multiple comparison method). Data are obtained from at least 3 independent experiments.</p

Unique maturation and activation status of nasal NK cells.

<p>a. Flow cytometry of CD3⁻NKp46⁺ cells from nasal passage, spleen, and lung, and double-stained with CD11b and CD27. Numbers in quadrants indicate the percentages of cells in each. Dot plots below shows percentage of CD27^highCD11b^low cells from nasal passage, spleen, and lung. b. Flow cytometry of CD3⁻NKp46⁺ cells from spleen, lung, and nasal passages with CD62L, CD69, and CD69/CD103. The numbers in the histograms indicate the percentage of positive cells. Solid line, specific antibody; dashed line, isotype-matched control antibody. Dot plots below shows the percentage of positively stained cells. Bar, mean; n.s.; not significant; *, <i>P</i> < 0.05**, <i>P</i> < 0.01 (Mann-Whitney <i>U</i> test with Ryan’s multiple comparison method). Data are obtained from at least 3 independent experiments.</p