crocodile mycoplasma crocodyli kirchhoff  (ATCC)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Licensed human natural killer cells aid dendritic cell maturation via TNFSF14/LIGHT

doi: 10.1073/pnas.1411072112

Figure Lengend Snippet: Gene expression profiling of tumor-stimulated human NK cells. (A) Primary human NK cells (isolated from healthy donors) were cocultured for 4 h with K562 tumor cells, and the R-NK and NR-NK cell populations were identified by staining for cell surface expression of CD56 (as a marker of NK cells) and CD107 (as a marker of NK cell degranulation). The R-NK and NR-NK populations were purified by FACS and used as a source of mRNA for cDNA synthesis and gene expression profiling using Illumina arrays. (Inset) CD107 display (x axis) vs. CD56 expression (y axis) on NK cells in the absence of tumor targets. (B) Venn diagram of the number of genes exhibiting a significant difference in expression between the R-NK and NR-NK populations (greater than 1.5-fold change; P < 0.05) of samples from a single donor and three pooled donors. A list of the 541 genes differentially expressed in both the single donor and pooled donor datasets is provided in Dataset S1. The array data are available from the Gene Expression Omnibus (ncbi.nlm.nih.gov/geo), under accession number GSE55977. The graph shows validation (using quantitative RT-PCR) of a selection of genes exhibiting differential expression in the array analysis (in order of increasing expression in the R-NK population, as determined by the array analysis). (C) Summary of differential expression between the R-NK and NR-NK samples. Selected genes exhibiting >1.5 fold upregulation (P < 0.05) in the R-NK cell fraction are shown according to the broad function of their encoded products. Functional details along with the gene identification number and supporting references are provided in Table S1. Genes with a defined AU rich element (ARE) in the 3' untranslated region (52, 53, 65, 66) are indicated (*), as are genes encoding molecules in the HVEM regulatory axis (**); TNFSF6 (FASL) has an ARE (67) and it is linked to the HVEM axis via its binding to DcR3(***). (D) The HVEM regulatory axis. Ligands (Top) include TNF superfamily (TNFSF) molecules (TNFSF2, TNFSF14, TNFSF15 and TNFSF6) as well as the immunoglobulin superfamily molecule CD160. Alternative names are also shown. The receptor molecules (Middle) all belong to the TNF receptor superfamily (TNFRSF). Soluble decoy receptor 3 (sDcR3; also known as TNFRSF6B) interacts with TNFSF14, TNFSF15 and TNFSF6 as shown, antagonizing their action. Both TNF (TNFSF2) and TNFSF14 (LIGHT) activate the transcription factor NF-κB (circled). This panel is based on information presented in published reviews (20, 21).

Article Snippet: These sorted NK cells were cocultured with iDCs at a 1:1 ratio for 48 h in either media alone or 12 μg/mL neutralizing anti-TNFSF14 antibody (goat polyclonal; R&D Systems), anti-TNF (goat polyclonal; R&D Systems), or normal goat IgG.

Techniques: Gene Expression, Isolation, Staining, Expressing, Marker, Purification, cDNA Synthesis, Biomarker Discovery, Quantitative RT-PCR, Selection, Quantitative Proteomics, Functional Assay, Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Licensed human natural killer cells aid dendritic cell maturation via TNFSF14/LIGHT

doi: 10.1073/pnas.1411072112

Figure Lengend Snippet: Tumor cell stimulation of NK cells induces production of TNFSF14. (A) Expression of cell surface molecules detected by gene expression profiling vs. CD107 display on unstimulated NK cells (No target) and NK cells stimulated with tumor cells (+K562). For the latter, the cell surface expression of molecules analyzed on the y axis is expressed as R/NR, the ratio of expression in the tumor-responding (CD107+) vs. tumor-nonresponding (CD107neg) populations (based on mean fluorescence intensity on the y axis; the corresponding gates are shown). These data are representative of three separate experiments performed. (B) Sustained expression of TNFSF14 (and CD69) in the continued presence of tumor cells. NK cells and K562 cells were cocultured for the times indicated, and cell surface TNFSF14 and CD69 were analyzed by flow cytometry. The values indicate the percentage of cells in each quadrant. cAb is an isotype control antibody for the anti-CD69 and anti-TNFSF14 staining. This experiment is representative of three separate experiments performed. (C) Induction of TNFSF14 expression requires de novo gene expression. NK cells alone (no target) or NK cells cocultured with K562 cells (+K562) were cultured for 5 h in the presence of 5 μM actinomycin D (ActD) to inhibit transcription or 50 μM cycloheximide (CHX) to inhibit translation (or with DMSO, the solvent for these compounds), and the cell surface expression of TNFSF14 and CD69 was analyzed by flow cytometry. The values indicate the percentage of cells in each quadrant. These data are representative of two separate experiments performed.

Article Snippet: These sorted NK cells were cocultured with iDCs at a 1:1 ratio for 48 h in either media alone or 12 μg/mL neutralizing anti-TNFSF14 antibody (goat polyclonal; R&D Systems), anti-TNF (goat polyclonal; R&D Systems), or normal goat IgG.

Techniques: Cell Stimulation, Expressing, Gene Expression, Fluorescence, Flow Cytometry, Control, Staining, Cell Culture, Solvent

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Licensed human natural killer cells aid dendritic cell maturation via TNFSF14/LIGHT

doi: 10.1073/pnas.1411072112

Figure Lengend Snippet: Induction of TNFSF14 by NK-cell activation receptor cross-linking. The cell surface expression of TNFSF14 and CD107 was analyzed on NK cells after coculture with P815 cells displaying antibodies against NK-cell activation receptors. P815 cells were loaded with antibodies against a single receptor or with combinations of receptors as indicated (or without antibody, labeled “Media”). K562 tumor cells were used in place of antibody-loaded P815 cells as a positive control for TNFSF14 expression and degranulation. The values indicate the percentage of cells in each quadrant. This experiment is representative of four separate experiments performed.

Article Snippet: These sorted NK cells were cocultured with iDCs at a 1:1 ratio for 48 h in either media alone or 12 μg/mL neutralizing anti-TNFSF14 antibody (goat polyclonal; R&D Systems), anti-TNF (goat polyclonal; R&D Systems), or normal goat IgG.

Techniques: Activation Assay, Expressing, Labeling, Positive Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Licensed human natural killer cells aid dendritic cell maturation via TNFSF14/LIGHT

doi: 10.1073/pnas.1411072112

Figure Lengend Snippet: Cytokine induction of immunomodulatory TNFSF14. (A) Expression of TNF or TNFSF14 in response to cytokine treatment or a combination of PMA and ionomycin (PMA/I), as indicated. NK cells (2.5 × 105) were cultured without stimulation (unstimulated) or in the presence of 50 U/mL IFN-I, 20 ng/mL IL-12 and IL-18, 300 U/mL IL-2, 30 ng/mL IL-15, or 50 ng/mL PMA plus 500 ng/mL ionomycin (PMA/I) for 8 h. Cells were stained for cell surface TNFSF14 expression or processed for intracellular staining for TNF. The values indicate the percentage of expressing cells. These data are representative of three donors for TNF and six donors for TNFSF14. (B) Release of soluble TNF or TNFSF14 by IL-2, IL-15, or PMA/I-treated NK cells. NK cells (2.5 × 105) were stimulated with the treatments shown in A for 24 h, and the supernatants were analyzed for TNF or TNFSF14 by ELISA (performed using at least three different donors). Media is the culture media without any added cells as a background control. Unstim., unstimulated. (C) Maturation of DCs in response to recombinant TNFSF14. The iDCs (differentiated from CD14+ monocytes) were left untreated or treated with recombinant TNF or TNFSF14 (100 ng/mL each) for 24 h and analyzed for cell surface markers of DC maturation, CD86, CD40, and HLA-DR using flow cytometry. (D, Left) Total of 1 × 105 IL-2–stimulated NK cells were applied to 3 × 105 iDCs for 15 h in the presence of antibodies against TNFSF14, TNF, or both cytokines (or cAb). The induction of CD86 on the iDCs was analyzed by flow cytometry, and the percentage of cells expressing CD86 is shown for each treatment. (D, Right) Summary of the data collected from five donors. Expression of CD86 on the DCs cocultured with IL-2–stimulated NK cells in the presence of cAb was assigned an expression level of 1. The reduction in expression of CD86 in the presence of the different anticytokine antibodies is indicated (*P < 0.05, **P < 0.005; paired Student’s t test).

Article Snippet: These sorted NK cells were cocultured with iDCs at a 1:1 ratio for 48 h in either media alone or 12 μg/mL neutralizing anti-TNFSF14 antibody (goat polyclonal; R&D Systems), anti-TNF (goat polyclonal; R&D Systems), or normal goat IgG.

Techniques: Expressing, Cell Culture, Staining, Enzyme-linked Immunosorbent Assay, Control, Recombinant, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Licensed human natural killer cells aid dendritic cell maturation via TNFSF14/LIGHT

doi: 10.1073/pnas.1411072112

Figure Lengend Snippet: TNFSF14 is produced by functionally licensed NK cells. Five donors were typed for KIR ligands and self-KIR molecules, as well as NKG2A expression, by flow cytometry. NK cells from these donors were then cocultured with K562 cells for 6 h, and cell surface TNFSF14 expression was analyzed on NK cells expressing none of these receptors, individual receptors, or combinations thereof (details of the gating strategy are provided in Fig. S3 and SI Materials and Methods). (A) Cell surface TNFSF14 expression on NK cells expressing no detectable NKG2A or self-reactive KIRs vs. NK cells expressing NKG2A and two self-reactive KIR molecules. The difference in TNFSF14 between these unlicensed and licensed NK cells is approximately 10-fold based on the mean fluorescence intensity of TNFSF14 expression. (B) Cell surface expression of TNFSF14 in response to tumor cell stimulation on NK cells expressing no (0 KIR), one (1KIR) or two (2KIR) self-reactive KIRs with or without NKG2A expression. The data show that any one of the inhibitory receptors (either NKG2A or KIR) confers a statistically significant effect on TNFSF14 expression (*P < 0.05, **P < 0.005; two-tailed Student’s t test). (C) Tumor-stimulated NK cells induce CD86 expression on iDCs in a TNFSF14-dependent manner. NK cells were cocultured with K562 tumor cells for 4 h, and the R-NK and NR-NK cells were isolated by FACS based on CD107 display alone. Samples from these sorted populations were reanalyzed for expression of cell surface TNFSF14 and CD107 molecules (Center, Post-sort NK), and the remaining cells were cocultured with iDCs (for 48 h at a ratio of 1:1) in the presence or absence of 10 μg/mL anti-TNFSF14 antibody or cAb. CD86 expression of the iDCs was analyzed by flow cytometry, and the percentage of CD86-expressing cells with the different treatments is shown. (Top) iDC analysis shows iDCs plus cAb without added NK cells. (D) Induction of cell surface CD86 expression on iDCs following coculture with NR-NK or R-NK cells in the presence of either cAb or anti-TNFSF14 antibody. These data are the fold change in mean fluorescence intensity of CD86 expression from three independent experiments (*P < 0.05, **P < 0.005**; two-tailed Student’s t test).

Article Snippet: These sorted NK cells were cocultured with iDCs at a 1:1 ratio for 48 h in either media alone or 12 μg/mL neutralizing anti-TNFSF14 antibody (goat polyclonal; R&D Systems), anti-TNF (goat polyclonal; R&D Systems), or normal goat IgG.

Techniques: Produced, Expressing, Flow Cytometry, Fluorescence, Cell Stimulation, Two Tailed Test, Isolation

Journal: PLoS ONE

Article Title: Approach to Analyze the Diversity of Myxobacteria in Soil by Semi-Nested PCR-Denaturing Gradient Gel Electrophoresis (DGGE) Based on Taxon-Specific Gene

doi: 10.1371/journal.pone.0108877

Figure Lengend Snippet: Strains used in this study.

Article Snippet: Myxococcus fulvus , DSM 16525 , Bacteria; Proteobacteria; Deltaproteobacteria; Myxococcales , DSMZ Germany.

Techniques: Bacteria

Journal: PLoS ONE

Article Title: Approach to Analyze the Diversity of Myxobacteria in Soil by Semi-Nested PCR-Denaturing Gradient Gel Electrophoresis (DGGE) Based on Taxon-Specific Gene

doi: 10.1371/journal.pone.0108877

Figure Lengend Snippet: A lane, 1, Archangium gephyra DSM 2261; lane 2, Corallococcus macrospores DSM 14697; lane 3, Corallococcus coralloides DSM 52499; lane 4, Myxococcus fulvus DSM 16525; lane 5, Myxococcus virescens DSM 2260; lane 6, Cystobacter minus DSM 14751; lane 7, Stigmatella erecta DSM 16858; lane 8, Byssovorax cruenta DSM 14533; B, Bands (1∼28) that were excised for sequence analysis are numbered.

Article Snippet: Myxococcus fulvus , DSM 16525 , Bacteria; Proteobacteria; Deltaproteobacteria; Myxococcales , DSMZ Germany.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Approach to Analyze the Diversity of Myxobacteria in Soil by Semi-Nested PCR-Denaturing Gradient Gel Electrophoresis (DGGE) Based on Taxon-Specific Gene

doi: 10.1371/journal.pone.0108877

Figure Lengend Snippet: Sequence similarity of excised DNA fragments.

Article Snippet: Myxococcus fulvus , DSM 16525 , Bacteria; Proteobacteria; Deltaproteobacteria; Myxococcales , DSMZ Germany.

Techniques: Sequencing