Inhibition of T_reg function by DC1 dendritic cells is not due to apoptosis.

<p>1.25×10⁵ sorted, purified CD4+CD25+ T cells were co-cultured with 1×10⁵ immature dendritic cells (<i>2A</i>) or DC1 dendritic cells (<i>2B</i>). Expression of the apoptotic markers Annexin-V and 7-AAD 24 hours later is shown. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074698#pone-0074698-g002" target="_blank">Figure 2C</a> summarizes the percent of cells expressing both markers (+/+), Annexin-V only (+/−), 7-AAD only (−/+), or neither marker (−/−). (iDC black, DC1 gray; <i>N = 4</i>).</p

DC1 dendritic cells inhibit T_regs directly.

<p>DC1 dendritic cells were generated as previously described. Medium from these cells was then harvested and combined 1∶1 with culture medium to create the “pretreatment” medium. CD4+CD25+ T cells or effectors were cultured in the pretreatment medium for 24 hours at a concentration of 3×10⁶ cells/mL. These “treated” cells were then harvested, washed, and used in cocultures as previously described. (<i>4A</i>) 2.5×10⁵ “treated” or “untreated” CFSE-labeled unfractionated responder lymphocytes were co-cultured with 1×10⁵ immature dendritic cells and 1.25×10⁵ sorted, purified CD4+CD25+ T cells for 5 days. CD4-positive responder cell proliferation is shown. (<i>4B</i>) 2.5×10⁵ CFSE-labeled unfractionated responder lymphocytes were co-cultured with 1×10⁵ immature dendritic cells and 1.25×10⁵ sorted, purified “treated” or “untreated” CD4+CD25+ T cells for 5 days. CD4-positive responder cell proliferation is shown. (<i>4C</i>) The number of mitoses per 10⁴ cells is summarized. In each case, data shown are representative of three separate experiments.</p

CD4+CD25+ T cells upregulate T-bet in the presence of DC1 dendritic cells.

<p>1.25×10⁵ CD4+CD25+ T cells were co-cultured with 1×10⁵ immature (<i>6A</i>) or DC1 (<i>6B,C</i>) dendritic cells. Neutralizing anti-IL-12 antibody (5 µg/mL) was included in some samples (<i>6C</i>). At 48 hours cells were harvested, permeabilized, and intracellular expression of T-bet and FoxP3 was detected by intracellular staining. Data shown are gated on CD4-positive cells and are representative of at least three separate experiments in each instance. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074698#pone-0074698-g006" target="_blank">Figure 6D</a> summarizes the percent of FoxP3+ cells that are T-bet positive at 48 hours for each group.</p

Inhibition of T_reg function by DC1 dendritic cells results from a soluble factor.

<p>(<i>3A</i>) 1.25×10⁵ sorted, purified CD4+CD25+ T cells were co-cultured with 2.5×10⁵ CFSE-labeled unfractionated responder lymphocytes and 1×10⁵ immature dendritic cells (solid line) or DC1 dendritic cells for 5 days (dashed line). Data shown are gated on CD4-positive cells and are representative of at least 10 experiments. (<i>3B</i>) 1.25×10⁵ T_regs were co-cultured with 2.5×10⁵ CFSE-labeled responders and 1×10⁵ immature dendritic cells for 5 days (solid line). In addition, 1×10⁵ DC1 dendritic cells were added to a transwell membrane placed in the culture well (dashed line). Data shown are gated on CD4-positive cells and are representative of 4 experiments. (<i>3C</i>) 1.25×10⁵ T_regs were co-cultured with 2.5×10⁵ CFSE-labeled responders alone for 5 days. 1×10⁵ DC1 dendritic cells were added to a transwell membrane placed in the culture well. Data shown are gated on CD4-positive cells (<i>N</i> = 2). (3<i>D</i>) The number of mitoses per 10⁴ cells is summarized for CD4-positive responder cells in the presence of T_regs and iDC alone, iDC with DC1 added to the Transwell membrane, and DC1 alone. (<i>3E&F</i>) 1.25×10⁵ T_regs were co-cultured with 2.5×10⁵ CFSE-labeled responders and 1×10⁵ DC1 dendritic cells in the presence (dashed line) or absence (solid line) of neutralizing anti-IL-12 (<i>3E</i>) or anti-IL-6 (<i>3F</i>) antibodies (5 µg/mL). Data shown are gated on CD4-positive cells and are representative of at least three experiments.</p

Suppressor CD4+CD25+ T cells secrete effector cytokines in the presence of DC1 dendritic cells.

<p>(<i>5A</i>) 2.5×10⁵ sorted CD4+CD25+ (T_reg) or CD4+CD25− (T_eff) T cells were combined with 2.0×10⁵ immature or DC1 dendritic cells. Supernatants were harvested at 5 days and ELISA was used to measure the amount of IFN-γ present in the supernatant. Data shown are the average of at least four experiments. (<i>5B</i>) At day 5, some culture samples were permeabilized and intracellular IFN-γ was detected by flow cytometry. (<i>5C&D</i>) 2.5×10⁵ sorted CD4+CD25+ T cells were cocultured with immature (<i>5C</i>) or DC1 (<i>5D</i>) dendritic cells; intracellular expression of FoxP3 and IFN-γ was detected in permeabilized cells 5 days later.</p

T_regs inhibit responder cell proliferation in the presence of immature but not DC1 dendritic cells.

<p>(<i>1A&B</i>) 2.5×10⁵ CFSE-labeled unfractionated responder lymphocytes were initially co-cultured with 1×10⁵ immature dendritic cells in the presence (dashed line) or absence (solid line) of 1.25×10⁵ sorted, purified CD4+CD25+ T cells for 5 days. Responder cell proliferation is shown for CD4-gated (<i>1A</i>) or CD8-gated (<i>1B</i>) T cells. Data shown are representative of at least 10 experiments. (<i>1C&1D</i>) 2.5×10⁵ CFSE-labeled unfractionated responders were co-cultured with 1.25×10⁵ sorted CD4+CD25+ T cells and 1×10⁵ immature (dashed line) or DC1 dendritic cells (solid line). Again responder cell proliferation is shown for CD4-gated (<i>1C</i>) or CD8-gated (<i>1D</i>) T cells. Data shown are representative of at least 10 experiments. (<i>1E</i>) 2.5×10⁵ CFSE-labeled unfractionated responders were co-cultured with 1×10⁵ dendritic cells matured using a conventional cytokine maturation cocktail (IL-1, IL-6, TNF-α, PGE-2) in the presence (dashed line) or absence (solid line) of 1.25×10⁵ sorted CD4+CD25+ T cells. Proliferation for CD4-gated T cells is shown (<i>N</i> = 4). (<i>1F</i>) Proliferation of CD4-positive responders in the presence of T_regs and immature dendritic cells treated briefly with LPS (15 minutes) prior to co-culture is shown (<i>N</i> = 3). (<i>1G</i>) A mathematical algorithm previously applied elsewhere was used to calculate the number of mitoses per 10⁴ responder CD4-gated T cells in the presence of iDC, DC1, or conventional cytokine maturation cocktail DC (CMDC).</p

Data_Sheet_1_Identification of key gene networks controlling polysaccharide accumulation in different tissues of Polygonatum cyrtonema Hua by integrating metabolic phenotypes and gene expression profiles.docx

Plant polysaccharides, a type of important bioactive compound, are involved in multiple plant defense mechanisms, and in particular polysaccharide-alleviated abiotic stress has been well studied. Polygonatum cyrtonema Hua (P. cyrtonema Hua) is a medicinal and edible perennial plant that is used in traditional Chinese medicine and is rich in polysaccharides. Previous studies suggested that sucrose might act as a precursor for polysaccharide biosynthesis. However, the role of sucrose metabolism and transport in mediating polysaccharide biosynthesis remains largely unknown in P. cyrtonema Hua. In this study, we investigated the contents of polysaccharides, sucrose, glucose, and fructose in the rhizome, stem, leaf, and flower tissues of P. cyrtonema Hua, and systemically identified the genes associated with the sucrose metabolism and transport and polysaccharide biosynthesis pathways. Our results showed that polysaccharides were mainly accumulated in rhizomes, leaves, and flowers. Besides, there was a positive correlation between sucrose and polysaccharide content, and a negative correlation between glucose and polysaccharide content in rhizome, stem, leaf, and flower tissues. Then, the transcriptomic analyses of different tissues were performed, and differentially expressed genes related to sucrose metabolism and transport, polysaccharide biosynthesis, and transcription factors were identified. The analyses of the gene expression patterns provided novel regulatory networks for the molecular basis of high accumulation of polysaccharides, especially in the rhizome tissue. Furthermore, our findings explored that polysaccharide accumulation was highly correlated with the expression levels of SUS, INV, SWEET, and PLST, which are mediated by bHLH, bZIP, ERF, ARF, C2H2, and other genes in different tissues of P. cyrtonema Hua. Herein, this study contributes to a comprehensive understanding of the transcriptional regulation of polysaccharide accumulation and provides information regarding valuable genes involved in the tolerance to abiotic stresses in P. cyrtonema Hua.</p

Data_Sheet_2_Identification of key gene networks controlling polysaccharide accumulation in different tissues of Polygonatum cyrtonema Hua by integrating metabolic phenotypes and gene expression profiles.xlsx

Plant polysaccharides, a type of important bioactive compound, are involved in multiple plant defense mechanisms, and in particular polysaccharide-alleviated abiotic stress has been well studied. Polygonatum cyrtonema Hua (P. cyrtonema Hua) is a medicinal and edible perennial plant that is used in traditional Chinese medicine and is rich in polysaccharides. Previous studies suggested that sucrose might act as a precursor for polysaccharide biosynthesis. However, the role of sucrose metabolism and transport in mediating polysaccharide biosynthesis remains largely unknown in P. cyrtonema Hua. In this study, we investigated the contents of polysaccharides, sucrose, glucose, and fructose in the rhizome, stem, leaf, and flower tissues of P. cyrtonema Hua, and systemically identified the genes associated with the sucrose metabolism and transport and polysaccharide biosynthesis pathways. Our results showed that polysaccharides were mainly accumulated in rhizomes, leaves, and flowers. Besides, there was a positive correlation between sucrose and polysaccharide content, and a negative correlation between glucose and polysaccharide content in rhizome, stem, leaf, and flower tissues. Then, the transcriptomic analyses of different tissues were performed, and differentially expressed genes related to sucrose metabolism and transport, polysaccharide biosynthesis, and transcription factors were identified. The analyses of the gene expression patterns provided novel regulatory networks for the molecular basis of high accumulation of polysaccharides, especially in the rhizome tissue. Furthermore, our findings explored that polysaccharide accumulation was highly correlated with the expression levels of SUS, INV, SWEET, and PLST, which are mediated by bHLH, bZIP, ERF, ARF, C2H2, and other genes in different tissues of P. cyrtonema Hua. Herein, this study contributes to a comprehensive understanding of the transcriptional regulation of polysaccharide accumulation and provides information regarding valuable genes involved in the tolerance to abiotic stresses in P. cyrtonema Hua.</p

Constructing robust 3D ionomer networks in the catalyst layer to achieve stable water electrolysis for green hydrogen production

The widespread application of proton exchange membrane water electrolyzers (PEMWEs) is hampered by insufficient lifetime caused by degradation of the anode catalyst layer (ACL). Here, an important degradation mechanism has been identified, attributed to poor mechanical stability causing the mass transfer channels to be blocked by ionomers under operating conditions. By using liquid-phase atomic force microscopy, we directly observed that the ionomers were randomly distributed (RD) in the ACL, which occupied the mass transfer channels due to swelling, creeping, and migration properties. Interestingly, we found that alternating treatments of the ACL in different water/temperature environments resulted in forming three-dimensional ionomer networks (3D INs) in the ACL, which increased the mechanical strength of microstructures by 3 times. Benefitting from the efficient and stable mass transfer channels, the lifetime was improved by 19 times. A low degradation rate of approximately 3.0 μV/h at 80 °C and a high current density of 2.0 A/cm2 was achieved on a 50 cm2 electrolyzer. These data demonstrated a forecasted lifetime of 80 000 h, approaching the 2026 DOE lifetime target. This work emphasizes the importance of the mechanical stability of the ACL and offers a general strategy for designing and developing a durable PEMWE.</p