pbmcs Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    Lonza peripheral blood mononuclear cells pbmcs
    Peripheral Blood Mononuclear Cells Pbmcs, supplied by Lonza, used in various techniques. Bioz Stars score: 97/100, based on 126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peripheral blood mononuclear cells pbmcs/product/Lonza
    Average 97 stars, based on 126 article reviews
    Price from $9.99 to $1999.99
    peripheral blood mononuclear cells pbmcs - by Bioz Stars, 2020-10
    97/100 stars
      Buy from Supplier

    99
    ATCC primary peripheral blood mononuclear cells pbmc normal human
    Tensor factorization to map model-predicted cytokine responses. A) Measured receptor abundance for ten <t>PBMC-derived</t> subpopulations. Points and error bars show geometric mean and standard deviation respectively (N = 4). Error bars for some points are too small to display. B-C) PCA scores (B) and loadings (C) of receptor abundance. Axis label percentages indicate percent variance explained. D) Schematic representation of CP decomposition. Model predictions are arranged in a cube depending upon the time, ligand treatment, and cell type being modeled. CP decomposition then helps to visualize this space. E) Percent variance reconstructed (R2X) versus the number of components used in non-negative CP decomposition. F-I) Component values versus time (F), cell type (G-H), or ligand stimulation (I). The variation explained by each component is the product of the component’s time, ligand, and cell type factorization. Ligand components with only negligible values (
    Primary Peripheral Blood Mononuclear Cells Pbmc Normal Human, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/primary peripheral blood mononuclear cells pbmc normal human/product/ATCC
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    primary peripheral blood mononuclear cells pbmc normal human - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    94
    Axis-Shield Diagnostics peripheral blood mononuclear cells pbmcs
    Lenalidomide maintenance therapy does not enhance MM-specific CD4 + T-cell responses Analysis of T-cell responses specific for the MAGE-C1 peptide SQSSPVSSFPSSTSS. ( A ) CD137 expression on CD4 + and CD8 + T cells from patient#02 at the pre <t>LEN</t> timepoint upon in vitro restimulation with the MAGE-C1 peptide. ( B ) <t>PBMCs</t> obtained at the pre LEN and the LEN timepoint were examined by IFN-γ ELISPOT to measure the T-cell response to the MAGE-C1 peptide. The bar graphs show the mean number of spots per million cells of four replicate wells and the error bars represent the standard deviation. A two-way ANOVA with Sidak’s multiple comparisons test was performed to evaluate statistical significant differences, ns: not significant, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.
    Peripheral Blood Mononuclear Cells Pbmcs, supplied by Axis-Shield Diagnostics, used in various techniques. Bioz Stars score: 94/100, based on 1342 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peripheral blood mononuclear cells pbmcs/product/Axis-Shield Diagnostics
    Average 94 stars, based on 1342 article reviews
    Price from $9.99 to $1999.99
    peripheral blood mononuclear cells pbmcs - by Bioz Stars, 2020-10
    94/100 stars
      Buy from Supplier

    94
    Thermo Fisher pbmcs
    Blockade of sialic-acid binding sites reveals BcR-dependent binding to influenza HA. A–B. Diffuse binding of rH1 to untreated human <t>PBMC.</t> Thawed <t>PBMCs</t> from an anonymous blood donor were first stained with Live/Dead then with an anti-CD20 mAb mixed with Alexa488-conjugated human serum albumin (PBS/HSA), or Alexa488-conjugated rH1 from the A/California/07/2009 strain (PBS/rH1), or a solution of sialopentasaccarides containing the Alexa488-conjugated rH1 (α-2,6-SPS/H1Cal). The strategy for gating on single live lymphocytes is shown in A; rH1 binding to CD20 negative (CD20 neg ) and CD20 + cells is shown in B. C. Blockade of sialic-acid binding sites reveals putative BcR-specifc binding of rH1. PBMCs from a single donor were split in 8 tubes (10 7 PBMC/each), 4 tubes were incubated with H3N2 mono-bulk subunit vaccine antigens from the A/Panama/2007/1999 strain (left dot plots), and 4 tubes with neuraminidase (right dot plots). Then the PBMCs were incubated with anti-CD20 and anti-CD27 mAbs and A488-HSA (upper plots), or A488-rH1 (lower plots). Shown is the distribution of rH1 binding on memory (CD27 + ) and naïve (CD27 neg ) B cells identified in the CD20 + gate. D. Specificity of rH1 binding. H1 + B-cells sorted from H3N2- (n = 1372) or neuraminidase- (Neur) (n = 2233) treated PBMCs were mixed with sorted autologous CD20 − cells in the ratio of 1∶200 and activated in vitro with CpG and IL-2 for 5 days. As controls, cultures of unsorted PBMCs, as well as of autologous CD20 neg cells mixed with H1-negative (H1 neg ) B cells sorted from neuraminidase-treated PBMCs (n = 30,000) in the ratio of 1∶10 were run for 5 days in the presence of CpG and IL-2. After 5 days, cells from each culture were harvested, washed, counted and assayed by ELISPOT to enumerate cells secreting IgG and H1N1-specific IgG. To facilitate comparisons between different cell cultures and across experiments depicted in different figures, the results are expressed as numbers of antibody secreting cells (ASC) normalized to 10 6 cultured cells assayed by ELISPOT.
    Pbmcs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 31889 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbmcs/product/Thermo Fisher
    Average 94 stars, based on 31889 article reviews
    Price from $9.99 to $1999.99
    pbmcs - by Bioz Stars, 2020-10
    94/100 stars
      Buy from Supplier

    94
    Becton Dickinson peripheral blood mononuclear cells pbmcs
    Measuring CTLA-4 and Fas expression by <t>CD4</t> + ,FOXP3 + T cells as well as apoptosis of CD4 + ,CD25 bright and CD25 − T cells in young and elderly humans. (A – D) <t>PBMCs</t> from young (n = 15) and elderly (n = 15) individuals were stained with Abs
    Peripheral Blood Mononuclear Cells Pbmcs, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 2072 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peripheral blood mononuclear cells pbmcs/product/Becton Dickinson
    Average 94 stars, based on 2072 article reviews
    Price from $9.99 to $1999.99
    peripheral blood mononuclear cells pbmcs - by Bioz Stars, 2020-10
    94/100 stars
      Buy from Supplier

    92
    Nycomed peripheral blood mononuclear cells pbmc
    CD69 expression in response to CMV antigens and to the influenza vaccine. <t>PBMCs</t> from elderly individuals were stimulated for 18 h and CD69 expression was evaluated by flow cytometry. a Representative dot plots of the frequency of CMV- and influenza vaccine-specific <t>CD4</t> T cells with and without NKG2D expression. Percentages of positive cells in this representative experiment are expressed in dot plots. b Histograms summarize the percentage of CD69-cells positive for CMV and influenza vaccine in the CD4+ CD28 null NKG2D+ ( black bars ) and CD4+ CD28 null NKG2D- subsets ( white bars ) obtained from 11 elderly donors. Paired t test was used to compare paired frequencies
    Peripheral Blood Mononuclear Cells Pbmc, supplied by Nycomed, used in various techniques. Bioz Stars score: 92/100, based on 502 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peripheral blood mononuclear cells pbmc/product/Nycomed
    Average 92 stars, based on 502 article reviews
    Price from $9.99 to $1999.99
    peripheral blood mononuclear cells pbmc - by Bioz Stars, 2020-10
    92/100 stars
      Buy from Supplier

    92
    Biochrom peripheral blood mononuclear cells pbmcs
    Single cell profiling identifies CD96 high and CD96 low subsets of Th9 cells. ( A – D ) Single cell gene expression analysis of CD44 + alloreactive T cells sorted from nonpolarized (w/o; n = 100), Th1 ( n = 67), and Th9 ( n = 113) polarized BALB/c cocultures. See Table S2 for a list of all genes measured. ( A ) Principal component analysis of gene expression in indicated Th subsets. ( B ) Nonsupervised hierarchical clustering analysis (Pearson’s correlation) comparing gene expression in Th subsets. Analysis includes all genes with detectable expression. ( C ) Box plots of most differentially expressed genes. Bonferroni-corrected ANOVA P values: Il9 ( P = 1.4 × 10 −126 ), Il2ra ( P = 1.4 × 10 −65 ), Cd83 ( P = 7.1 × 10 −64 ), Il4ra ( P = 1.2 × 10 −52 ), Gata3 ( P = 3.6 × 10 −47 ), Il1r1 ( P = 5.5 × 10 −47 ), Ccr4 ( P = 2.2 × 10 −46 ), and Cd28 ( P = 2.3 × 10 −43 ). ( D ) Nonsupervised hierarchical clustering analysis (complete linkage method) of gene expression in CD44 + T cells sorted from alloreactive BALB/c Th9 cultures. Displayed genes contributed to clustering. Frame indicates a cluster of Th9 cells characterized by high Cd96 expression. ( E ) Contour plots of surface CD96 expression in CD44 + polarized alloreactive BALB/c T cells, including fluorescence minus one (FMO) staining control (Ctrl). ( F ) Comparison of CD96 expression levels in IL-9 + and IFN-γ + human <t>CD4</t> + T cells. <t>PBMCs</t> from blood of 10 healthy individuals were stimulated ex vivo with Staphylococcus enterotoxin B (SEB) for 24 h. A one-tailed Wilcoxon matched-pairs signed rank test was applied. gMFI, geometric mean fluorescence intensity.
    Peripheral Blood Mononuclear Cells Pbmcs, supplied by Biochrom, used in various techniques. Bioz Stars score: 92/100, based on 407 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peripheral blood mononuclear cells pbmcs/product/Biochrom
    Average 92 stars, based on 407 article reviews
    Price from $9.99 to $1999.99
    peripheral blood mononuclear cells pbmcs - by Bioz Stars, 2020-10
    92/100 stars
      Buy from Supplier

    89
    Ficoll-Paque Pharmacia peripheral blood mononuclear cell pbmc
    Semi-quantitative <t>PCR</t> analysis of BLV pol detects differences in proviral load between BLV SGV and BLV-rabbits. <t>PBMC</t> were harvested at month indicated after challenge with 1 × 10 8 FLK/BLV cells from indicated rabbits that had been infected with BLV SGV for 24 months or from age-matched naïve controls. DNA from 50,000 PMBC was subjected to nested PCR and gel electrophoresis and stained with ethidium bromide. The position of the 591-bp amplicon is designated by the labeled arrow. In parallel, DNA from 20,000 PBMC was subjected to PCR with primers complementary to β- actin . The position of the 594-bp amplicon is designated by the labeled arrow and the signals were quantified by densitometry.
    Peripheral Blood Mononuclear Cell Pbmc, supplied by Ficoll-Paque Pharmacia, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peripheral blood mononuclear cell pbmc/product/Ficoll-Paque Pharmacia
    Average 89 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    peripheral blood mononuclear cell pbmc - by Bioz Stars, 2020-10
    89/100 stars
      Buy from Supplier

    96
    Millipore pbmcs
    The total number of circulating fibrocytes is unaffected by the leukocyte isolation strategy used. a Total number of circulating fibrocytes (defined as CD45 + lin − CD15 − CXCR4 + Col-1 + cells) per ml blood using two common leukocyte isolation techniques, e.g. the simple Pasteur pipette tube technique to isolate all white blood cells and <t>Ficoll</t> separation technique to isolate <t>PBMCs.</t> For this experiment we analyzed paired total white blood cells and PBMCs on the same day as blood withdrawal of 9 patients (4 IPF patients (black) and 5 PH patients (purple)) and 5 healthy controls (green). b Percentage of fibrocytes (of CD45 + cells) in the same patients after isolating all white blood cells (gray) and PBMCs (black). Data are depicted as median and interquartile
    Pbmcs, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 20755 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbmcs/product/Millipore
    Average 96 stars, based on 20755 article reviews
    Price from $9.99 to $1999.99
    pbmcs - by Bioz Stars, 2020-10
    96/100 stars
      Buy from Supplier

    93
    Becton Dickinson human pbmcs
    <t>EAT-2</t> over-expression enhances human DC maturation. Human <t>PBMCs</t> were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-EAT2 or rAd5-Null. At 72 hpi, cells were stained for surface expression of CD86 (A), HLA-DR (B), CD80 (C) and CD83 (D) and FACS analysis was performed. Data are representative of at least three independent experiments from three different blood donors with similar results. Samples were plated in quadruplicates. Bars represent mean ± SD. Statistical analysis was completed using a one way ANOVA with a Student–Newman–Keuls post-hoc test. P
    Human Pbmcs, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 603 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human pbmcs/product/Becton Dickinson
    Average 93 stars, based on 603 article reviews
    Price from $9.99 to $1999.99
    human pbmcs - by Bioz Stars, 2020-10
    93/100 stars
      Buy from Supplier

    93
    Becton Dickinson blood mononuclear cells pbmcs
    Frequency and molecular expression of pDC in IT, CHB and AHB groups. (a) AHB group had significantly higher <t>PBMC</t> percent in peripheral blood leukocytes, compared with CHB or IT group. (b) Frequency of pDC in PBMC decreased in AHB and CHB groups, compared with IT group. (c) The difference in CD86+ pDC frequency among three groups was not significant. (d) The counts of CD86 molecular expressed on surface of pDC in CHB group was higher, compared with AHB and IT groups. IT group: HBeAg-positive chronic hepatitis B virus infected patients in immune tolerance phase; CHB group: HBeAg-positive chronic hepatitis B infected patients; AHB group: Acute hepatitis B virus infected patients; PBMC: Peripheral blood mononuclear cells; pDC: <t>Plasmacytoid</t> dendritic cell; CD86: Cluster of differentiation antigen 86.
    Blood Mononuclear Cells Pbmcs, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 339 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/blood mononuclear cells pbmcs/product/Becton Dickinson
    Average 93 stars, based on 339 article reviews
    Price from $9.99 to $1999.99
    blood mononuclear cells pbmcs - by Bioz Stars, 2020-10
    93/100 stars
      Buy from Supplier

    92
    Valiant peripheral blood mononuclear cells pbmcs
    Exosomes from tumor patients with increased membrane-associated HSP72 and HSP105 induced <t>DCs</t> to promote tumor cell invasion via IL-6 in vitro . (A) After adsorption onto latex beads, HSP72 and HSP105 on EXO-PT and EXO-TT were detected by flow cytometry. RFI was calculated by dividing mean fluorescence intensity of samples staining with detection Abs by that of sample staining with ISO Abs (n = 7). (B) Human DCs were induced from <t>PBMCs</t> and stimulated with 5 μg/ml of EXO-PT or EXO-TT from breast tumor tissues for 6 h. The IL-6 production was measured by ELISA (n = 3). (C) MDA-MB-435S cells were cultured in supernatant from PBS-, EXO-PT- or EXO-TT (5 μg/ml for 6 h)-stimulated DCs with or without 30 μg/ml of anti-IL-6 or ISO mAbs for 24 h. Then, their invasive ability was measured using an in vitro invasive assay, and the cells on the bottom of the Transwell filter were imaged and quantified (Magnification: 200 ×). (D) The results of (C) were statistically analyzed (n = 5). (E, F) After adsorption onto latex beads, HSP72 and HSP105 on exosomes from sera of breast tumor patients or healthy controls were detected by flow cytometry. One representative result of 12 is shown (E). RFI was calculated and statistically analyzed (F). (B, D) The results are shown as the mean ± SEM of 3 independent experiments. (C) One representative of 3 independent experiments is shown. P values were generated by 2-tail student's t -test in A, F, and by one-way ANOVA, followed by a Tukey-Kramer multiple comparison test in B, D; *** p
    Peripheral Blood Mononuclear Cells Pbmcs, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peripheral blood mononuclear cells pbmcs/product/Valiant
    Average 92 stars, based on 193 article reviews
    Price from $9.99 to $1999.99
    peripheral blood mononuclear cells pbmcs - by Bioz Stars, 2020-10
    92/100 stars
      Buy from Supplier

    93
    Corning Life Sciences pbmc
    Unchanged healthy donor NK cell degranulation in response to GAD65 AA 114–122 peptide-pulsed <t>APCs.</t> Degranulation of CD3 - CD56 + ILT2 + NK cells of <t>PBMC</t> from HD patients, expanded with GAD65 AA 114–122 peptide, measured as CD107a cell-surface expression following stimulation with APCs, either left unpulsed or GAD65 AA 114–122 peptide-pulsed. A representative experiment out of two performed is shown. K562 cells were used as positive control. The percentage of CD3 - CD56 + CD107a + NK cells (upper panel) and of CD3 - CD56 + CD107a + ILT2 + (lower panel) is indicated for each condition.
    Pbmc, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 93/100, based on 688 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbmc/product/Corning Life Sciences
    Average 93 stars, based on 688 article reviews
    Price from $9.99 to $1999.99
    pbmc - by Bioz Stars, 2020-10
    93/100 stars
      Buy from Supplier

    92
    AllCells LLC pbmcs
    Inference and effect of barcode multiplets in single-cell ATAC-seq data. (a) Default t-SNE depiction of public <t>scATAC-seq</t> <t>PBMC</t> 5k dataset. Colors represent cluster annotations from the automated CellRanger output. (b) Quantification of barcodes affected by barcode multiplets for the same dataset (identified by bap). (c) Depiction of two multiplets each composed of 9 oligonucleotide barcodes. Barcodes in each multiplet share a long common subsequence, denoted in black. (d) Visualization of two barcode multiplets from (c) in t-SNE coordinates. (e) Visualization of all implicated barcode multiplets from this dataset. The zoomed panel shows a small group of cells affected by five multiplets, indicated by color. (f) Empirical distribution of the mean restricted longest common subsequence (rLCS) per multiplet. A cutoff of 6 was used to determine either of the two classes of barcode multiplets. (g) Percent difference of the mean log2 fragments between pairs of barcodes within a multiplet. The reported p-value is from a two-sided Kolmogorov–Smirnov test. Boxplots: center line, median; box limits, first and third quartiles; whiskers, 1.5x interquartile range. (h) Overall rates of barcode multiplets from additional scATAC-seq data comparing v1.0 and v1.1 (NextGEM) chip designs.
    Pbmcs, supplied by AllCells LLC, used in various techniques. Bioz Stars score: 92/100, based on 153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbmcs/product/AllCells LLC
    Average 92 stars, based on 153 article reviews
    Price from $9.99 to $1999.99
    pbmcs - by Bioz Stars, 2020-10
    92/100 stars
      Buy from Supplier

    93
    Becton Dickinson pbmc isolation
    ( a ) p24 viral antigen levels 10 days post-infection in the supernatant of <t>HIV-1</t> infected PBMCs treated with TIZ (10 μg/mL) or RM-4848 (10 μg/mL). Results obtained with <t>PBMC</t> of 20 different healthy donors are shown. Median values and statistical significance is presented. ( b ) Inhibition (%) of viral replication in PBMC of 20 different healthy donors infected in vitro with HIV-1 in the presence of TIZ- (10 μg/mL) or RM-4848- (10 μg/mL). Median values and statistical significance is presented. ( c ) p24 viral antigen levels in PBMCs infected with HIV-1 in the presence of RM-4848 throughout infection (RM-4848)(10 μg/mL) or in medium alone (no treatment). In addition to these two situations, results of 3 other experimental conditions are shown: 1) PBMCs treated with RM-4848 before being infected (pre-infection); 2) RM-4848 present during the 24 hours infection period (during infection) and then washed away; 3) RM-4848 added 24 hours after infection (post infection). Mean values ± standard errors are shown.
    Pbmc Isolation, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 179 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbmc isolation/product/Becton Dickinson
    Average 93 stars, based on 179 article reviews
    Price from $9.99 to $1999.99
    pbmc isolation - by Bioz Stars, 2020-10
    93/100 stars
      Buy from Supplier

    99
    Millipore blood mononuclear cells pbmcs
    ( a ) p24 viral antigen levels 10 days post-infection in the supernatant of <t>HIV-1</t> infected PBMCs treated with TIZ (10 μg/mL) or RM-4848 (10 μg/mL). Results obtained with <t>PBMC</t> of 20 different healthy donors are shown. Median values and statistical significance is presented. ( b ) Inhibition (%) of viral replication in PBMC of 20 different healthy donors infected in vitro with HIV-1 in the presence of TIZ- (10 μg/mL) or RM-4848- (10 μg/mL). Median values and statistical significance is presented. ( c ) p24 viral antigen levels in PBMCs infected with HIV-1 in the presence of RM-4848 throughout infection (RM-4848)(10 μg/mL) or in medium alone (no treatment). In addition to these two situations, results of 3 other experimental conditions are shown: 1) PBMCs treated with RM-4848 before being infected (pre-infection); 2) RM-4848 present during the 24 hours infection period (during infection) and then washed away; 3) RM-4848 added 24 hours after infection (post infection). Mean values ± standard errors are shown.
    Blood Mononuclear Cells Pbmcs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 524 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/blood mononuclear cells pbmcs/product/Millipore
    Average 99 stars, based on 524 article reviews
    Price from $9.99 to $1999.99
    blood mononuclear cells pbmcs - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    92
    PAA Laboratories peripheral blood mononuclear cells pbmcs
    ( a ) p24 viral antigen levels 10 days post-infection in the supernatant of <t>HIV-1</t> infected PBMCs treated with TIZ (10 μg/mL) or RM-4848 (10 μg/mL). Results obtained with <t>PBMC</t> of 20 different healthy donors are shown. Median values and statistical significance is presented. ( b ) Inhibition (%) of viral replication in PBMC of 20 different healthy donors infected in vitro with HIV-1 in the presence of TIZ- (10 μg/mL) or RM-4848- (10 μg/mL). Median values and statistical significance is presented. ( c ) p24 viral antigen levels in PBMCs infected with HIV-1 in the presence of RM-4848 throughout infection (RM-4848)(10 μg/mL) or in medium alone (no treatment). In addition to these two situations, results of 3 other experimental conditions are shown: 1) PBMCs treated with RM-4848 before being infected (pre-infection); 2) RM-4848 present during the 24 hours infection period (during infection) and then washed away; 3) RM-4848 added 24 hours after infection (post infection). Mean values ± standard errors are shown.
    Peripheral Blood Mononuclear Cells Pbmcs, supplied by PAA Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peripheral blood mononuclear cells pbmcs/product/PAA Laboratories
    Average 92 stars, based on 71 article reviews
    Price from $9.99 to $1999.99
    peripheral blood mononuclear cells pbmcs - by Bioz Stars, 2020-10
    92/100 stars
      Buy from Supplier

    93
    Greiner Bio peripheral blood mononuclear cells pbmcs
    ( a ) p24 viral antigen levels 10 days post-infection in the supernatant of <t>HIV-1</t> infected PBMCs treated with TIZ (10 μg/mL) or RM-4848 (10 μg/mL). Results obtained with <t>PBMC</t> of 20 different healthy donors are shown. Median values and statistical significance is presented. ( b ) Inhibition (%) of viral replication in PBMC of 20 different healthy donors infected in vitro with HIV-1 in the presence of TIZ- (10 μg/mL) or RM-4848- (10 μg/mL). Median values and statistical significance is presented. ( c ) p24 viral antigen levels in PBMCs infected with HIV-1 in the presence of RM-4848 throughout infection (RM-4848)(10 μg/mL) or in medium alone (no treatment). In addition to these two situations, results of 3 other experimental conditions are shown: 1) PBMCs treated with RM-4848 before being infected (pre-infection); 2) RM-4848 present during the 24 hours infection period (during infection) and then washed away; 3) RM-4848 added 24 hours after infection (post infection). Mean values ± standard errors are shown.
    Peripheral Blood Mononuclear Cells Pbmcs, supplied by Greiner Bio, used in various techniques. Bioz Stars score: 93/100, based on 230 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peripheral blood mononuclear cells pbmcs/product/Greiner Bio
    Average 93 stars, based on 230 article reviews
    Price from $9.99 to $1999.99
    peripheral blood mononuclear cells pbmcs - by Bioz Stars, 2020-10
    93/100 stars
      Buy from Supplier

    91
    Becton Dickinson flow cytometry pbmcs
    Rituximab ( RTX ) leads to further phenotypical changes in CD56 dim natural killer ( NK ) cells. Freshly isolated peripheral blood mononuclear cells <t>(PBMCs)</t> were incubated with (+) or without ( w/o ) RTX overnight. The next day PBMCs were stained with a mixture of antibodies (anti-CD3 PE-Dazzle, anti-CD56 Bv421 and anti-CD16 FITC and CD69/CD137 PE) and analyzed by flow <t>cytometry.</t> NK cells were identified as CD3-CD56+ cells in the lymphocyte gate and fluorescence intensity of PE on CD56 dim NK cells was measured. a CD69 PE. Histogram shows one representative donor; gray incubation + RTX, black w/o RTX. Left diagram mean CD69 PE fluorescence intensity in samples from 13 donors. Statistical significance was determined with the Wilcoxon signed rank test (p = 0.0002). Right diagram Δ % in CD56 dim NK was determined by (CD69-positive CD56 dim NK cells after incubation + RTX) - (CD69-positive CD56 dim NK cells after incubation w/o RTX); bar median. b CD137(41BB)-PE; same donor, layout and abbreviations as in a ; n = 11 donors; p = 0.001
    Flow Cytometry Pbmcs, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 254 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flow cytometry pbmcs/product/Becton Dickinson
    Average 91 stars, based on 254 article reviews
    Price from $9.99 to $1999.99
    flow cytometry pbmcs - by Bioz Stars, 2020-10
    91/100 stars
      Buy from Supplier

    92
    Cellular Technology Ltd pbmcs
    Suppression of <t>CTL</t> responses to the AAV capsid in vitro mediated by Tregitopes is contact mediated and results in anergy of effector cell . ( a ) Transwell experiment outline. Cells were placed in a transwell chamber in which the upper and lower chambers were separated by a permeable membrane (drawing). The table shows the different conditions tested in the transwell; in each of the transwell chambers untouched <t>PBMC</t> or PBMC depleted of CD4 + (CD4 neg CD8 + ) or CD8 + (CD4 + CD8 neg ) T cells were plated and restimulated with either AAV-only or AAV+hTreg167 peptides. After restimulation, PBMC or CD4 neg CD8 + cells were harvested from the transwell and used in a CTL assay against target cells loaded with the AAV-derived MHC I epitope VPYGYLTL at a Teff:target ratio 10:1. Results are expressed as % cytotoxicity compared to a max cytotoxicity (cells treated with 10% SDS) after background subtraction. ( b ) CTL assay in which target cells were transduced with an AAV vector at increasing MOIs and then incubated with HLA-matched effector cells. Teff cells were obtained by restimulating PBMC in vitro with the AAV capsid MHC I peptide VPQYGYLTL alone (AAV, black line), with AAV+Tregitope 167 (AAV+hTreg167, dashed black line), with AAV+hTreg167 followed by CD4 + T cell depletion (AAV+hTreg167 [CD4 neg ], gray line circles), or with AAV+hTreg167 followed by CD4 + T cell depletion and incubation with 10ng/ml IL-2 overnight (AAV+hTreg167 [CD4 neg + IL-2], gray line triangles). Teff:target ratio 10:1. Results are expressed as % cytotoxicity compared to a max cytotoxicity (cells treated with 10% SDS) after background subtraction. Experiments shown were repeated at least twice. Error bars represent SEM.
    Pbmcs, supplied by Cellular Technology Ltd, used in various techniques. Bioz Stars score: 92/100, based on 199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbmcs/product/Cellular Technology Ltd
    Average 92 stars, based on 199 article reviews
    Price from $9.99 to $1999.99
    pbmcs - by Bioz Stars, 2020-10
    92/100 stars
      Buy from Supplier

    93
    Axis-Shield Diagnostics blood mononuclear cells pbmcs
    The IL-27 rs153109 A > G polymorphism enhanced pro-inflammatory cytokine release of the human peripheral blood mononuclear cells <t>(PBMCs).</t> The PBMCs of 50 healthy volunteers were incubated in vitro and stimulated with 500 ng/mL lipolysaccharide (LPS) for 8 h. Then the supernatant concentration of TNF-α, IL-6 and IL-1β in groups with different rs153109 genotypes were measured ( a - f ). The horizontal line indicates the mean expression level within each genotype group. The error bar represents standard error of the mean
    Blood Mononuclear Cells Pbmcs, supplied by Axis-Shield Diagnostics, used in various techniques. Bioz Stars score: 93/100, based on 126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/blood mononuclear cells pbmcs/product/Axis-Shield Diagnostics
    Average 93 stars, based on 126 article reviews
    Price from $9.99 to $1999.99
    blood mononuclear cells pbmcs - by Bioz Stars, 2020-10
    93/100 stars
      Buy from Supplier

    Image Search Results


    Tensor factorization to map model-predicted cytokine responses. A) Measured receptor abundance for ten PBMC-derived subpopulations. Points and error bars show geometric mean and standard deviation respectively (N = 4). Error bars for some points are too small to display. B-C) PCA scores (B) and loadings (C) of receptor abundance. Axis label percentages indicate percent variance explained. D) Schematic representation of CP decomposition. Model predictions are arranged in a cube depending upon the time, ligand treatment, and cell type being modeled. CP decomposition then helps to visualize this space. E) Percent variance reconstructed (R2X) versus the number of components used in non-negative CP decomposition. F-I) Component values versus time (F), cell type (G-H), or ligand stimulation (I). The variation explained by each component is the product of the component’s time, ligand, and cell type factorization. Ligand components with only negligible values (

    Journal: bioRxiv

    Article Title: Modeling Cell-Specific Dynamics and Regulation of the Common Gamma Chain Cytokines

    doi: 10.1101/778894

    Figure Lengend Snippet: Tensor factorization to map model-predicted cytokine responses. A) Measured receptor abundance for ten PBMC-derived subpopulations. Points and error bars show geometric mean and standard deviation respectively (N = 4). Error bars for some points are too small to display. B-C) PCA scores (B) and loadings (C) of receptor abundance. Axis label percentages indicate percent variance explained. D) Schematic representation of CP decomposition. Model predictions are arranged in a cube depending upon the time, ligand treatment, and cell type being modeled. CP decomposition then helps to visualize this space. E) Percent variance reconstructed (R2X) versus the number of components used in non-negative CP decomposition. F-I) Component values versus time (F), cell type (G-H), or ligand stimulation (I). The variation explained by each component is the product of the component’s time, ligand, and cell type factorization. Ligand components with only negligible values (

    Article Snippet: Receptor abundance quantitation Cryopreserved PBMCs (ATCC, PCS-800-011, lot#81115172) were thawed to room temperature and slowly diluted with 9 mL pre-warmed RPMI-1640 medium (Gibco, 11875-093) supplemented with 10% fetal bovine serum (FBS, Seradigm, 1500-500, lot#322B15).

    Techniques: Derivative Assay, Standard Deviation

    Receptor quantification and gating of PBMC-derived immune cell types. A) Preliminary gating for single lyphocytes. B) Example staining for CD122 (red), the corresponding isotype control (blue), and unstained cells (black). C) Gating for live T helper and T regulatory cells during receptor quantification. D) Live cell NK cell gating. E) Live cell CD8+ T cell gating. F) Gating for fixed T helper and T regulatory cells during pSTAT5 quantification. G) Fixed CD8+ T cell and NK cell gating.

    Journal: bioRxiv

    Article Title: Modeling Cell-Specific Dynamics and Regulation of the Common Gamma Chain Cytokines

    doi: 10.1101/778894

    Figure Lengend Snippet: Receptor quantification and gating of PBMC-derived immune cell types. A) Preliminary gating for single lyphocytes. B) Example staining for CD122 (red), the corresponding isotype control (blue), and unstained cells (black). C) Gating for live T helper and T regulatory cells during receptor quantification. D) Live cell NK cell gating. E) Live cell CD8+ T cell gating. F) Gating for fixed T helper and T regulatory cells during pSTAT5 quantification. G) Fixed CD8+ T cell and NK cell gating.

    Article Snippet: Receptor abundance quantitation Cryopreserved PBMCs (ATCC, PCS-800-011, lot#81115172) were thawed to room temperature and slowly diluted with 9 mL pre-warmed RPMI-1640 medium (Gibco, 11875-093) supplemented with 10% fetal bovine serum (FBS, Seradigm, 1500-500, lot#322B15).

    Techniques: Derivative Assay, Staining

    Model accurately predicts cell type-specific response across a panel of PBMC-derived cell types. A) Comparison of two replicates measuring pSTAT5 response to a dose-response of IL-2/-15, time course, and panel of PBMC-derived cell types. B) Both experimentally-derived and model-predicted EC 50 s of dose response across IL-2/-15 and all 10 cell types. EC 50 s are shown for 1 hr time point. C) Pearson correlation coefficients between model prediction and experimental measurements for all 10 cell populations (full data shown in Fig. S5 ). D–I) pSTAT5 response to IL-2 (D-F) or IL-15 (G-I) dose responses in NK, CD8+, and T reg cells.

    Journal: bioRxiv

    Article Title: Modeling Cell-Specific Dynamics and Regulation of the Common Gamma Chain Cytokines

    doi: 10.1101/778894

    Figure Lengend Snippet: Model accurately predicts cell type-specific response across a panel of PBMC-derived cell types. A) Comparison of two replicates measuring pSTAT5 response to a dose-response of IL-2/-15, time course, and panel of PBMC-derived cell types. B) Both experimentally-derived and model-predicted EC 50 s of dose response across IL-2/-15 and all 10 cell types. EC 50 s are shown for 1 hr time point. C) Pearson correlation coefficients between model prediction and experimental measurements for all 10 cell populations (full data shown in Fig. S5 ). D–I) pSTAT5 response to IL-2 (D-F) or IL-15 (G-I) dose responses in NK, CD8+, and T reg cells.

    Article Snippet: Receptor abundance quantitation Cryopreserved PBMCs (ATCC, PCS-800-011, lot#81115172) were thawed to room temperature and slowly diluted with 9 mL pre-warmed RPMI-1640 medium (Gibco, 11875-093) supplemented with 10% fetal bovine serum (FBS, Seradigm, 1500-500, lot#322B15).

    Techniques: Derivative Assay

    A reaction model captures cytokine-cytokine interactions. A) Schematic of IL-4 and IL-7 receptor complexes competing for γ c and generating distinct pSTAT signals. B-C) Fitting model to experimental data. Experimental measurements are denoted by triangles. Shaded areas represent the 25-75% and 10-90% confidence intervals of model predictions. pSTAT5 and pSTAT6 were measured for IL-7 and IL-4 experiments, respectively. B) Singlecytokine pSTAT dose-response measurements for 10 min of exposure to IL-4 and IL-7. C) Percent inhibition of the second cytokine’s pSTAT response in a dual-cytokine dose-response experiment. Human PBMC-derived T cells (CD4 + TCR + CCR7 high ) were pretreated with various concentrations of one cytokine for 10 min before being stimulated with a fixed concentration (50 pg/mL IL-7 or 100 pg/mL IL-4) of the other cytokine for an additional 10 min. D) Model predictions for percent inhibition of the second cytokine’s pSTAT response in a dual-cytokine dose-response experiment with the assumption that active species are endocytosed at the same rate as inactive species (k endo,a = k endo ). E) Model predictions for percent of γ c on the cell surface when exposed to 100 pg/mL of either IL-7 or IL-4 for 100 min. F) Violin plot of K a values obtained via posterior distributions of k fwd / k rev for krev parameters corresponding to different complexes competing for the common γ c ( Fig. 1A ). G–I) Posterior distributions from fitting to data. Scaling constants C 5 and C 6 have units of # × cell −1 , k fwd has units of cell × # −1 × min −1 , and f sort is unitless

    Journal: bioRxiv

    Article Title: Modeling Cell-Specific Dynamics and Regulation of the Common Gamma Chain Cytokines

    doi: 10.1101/778894

    Figure Lengend Snippet: A reaction model captures cytokine-cytokine interactions. A) Schematic of IL-4 and IL-7 receptor complexes competing for γ c and generating distinct pSTAT signals. B-C) Fitting model to experimental data. Experimental measurements are denoted by triangles. Shaded areas represent the 25-75% and 10-90% confidence intervals of model predictions. pSTAT5 and pSTAT6 were measured for IL-7 and IL-4 experiments, respectively. B) Singlecytokine pSTAT dose-response measurements for 10 min of exposure to IL-4 and IL-7. C) Percent inhibition of the second cytokine’s pSTAT response in a dual-cytokine dose-response experiment. Human PBMC-derived T cells (CD4 + TCR + CCR7 high ) were pretreated with various concentrations of one cytokine for 10 min before being stimulated with a fixed concentration (50 pg/mL IL-7 or 100 pg/mL IL-4) of the other cytokine for an additional 10 min. D) Model predictions for percent inhibition of the second cytokine’s pSTAT response in a dual-cytokine dose-response experiment with the assumption that active species are endocytosed at the same rate as inactive species (k endo,a = k endo ). E) Model predictions for percent of γ c on the cell surface when exposed to 100 pg/mL of either IL-7 or IL-4 for 100 min. F) Violin plot of K a values obtained via posterior distributions of k fwd / k rev for krev parameters corresponding to different complexes competing for the common γ c ( Fig. 1A ). G–I) Posterior distributions from fitting to data. Scaling constants C 5 and C 6 have units of # × cell −1 , k fwd has units of cell × # −1 × min −1 , and f sort is unitless

    Article Snippet: Receptor abundance quantitation Cryopreserved PBMCs (ATCC, PCS-800-011, lot#81115172) were thawed to room temperature and slowly diluted with 9 mL pre-warmed RPMI-1640 medium (Gibco, 11875-093) supplemented with 10% fetal bovine serum (FBS, Seradigm, 1500-500, lot#322B15).

    Techniques: Inhibition, Derivative Assay, Concentration Assay

    Lenalidomide maintenance therapy does not enhance MM-specific CD4 + T-cell responses Analysis of T-cell responses specific for the MAGE-C1 peptide SQSSPVSSFPSSTSS. ( A ) CD137 expression on CD4 + and CD8 + T cells from patient#02 at the pre LEN timepoint upon in vitro restimulation with the MAGE-C1 peptide. ( B ) PBMCs obtained at the pre LEN and the LEN timepoint were examined by IFN-γ ELISPOT to measure the T-cell response to the MAGE-C1 peptide. The bar graphs show the mean number of spots per million cells of four replicate wells and the error bars represent the standard deviation. A two-way ANOVA with Sidak’s multiple comparisons test was performed to evaluate statistical significant differences, ns: not significant, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.

    Journal: Oncotarget

    Article Title: Impact of lenalidomide maintenance on the immune environment of multiple myeloma patients with low tumor burden after autologous stem cell transplantation

    doi: 10.18632/oncotarget.24944

    Figure Lengend Snippet: Lenalidomide maintenance therapy does not enhance MM-specific CD4 + T-cell responses Analysis of T-cell responses specific for the MAGE-C1 peptide SQSSPVSSFPSSTSS. ( A ) CD137 expression on CD4 + and CD8 + T cells from patient#02 at the pre LEN timepoint upon in vitro restimulation with the MAGE-C1 peptide. ( B ) PBMCs obtained at the pre LEN and the LEN timepoint were examined by IFN-γ ELISPOT to measure the T-cell response to the MAGE-C1 peptide. The bar graphs show the mean number of spots per million cells of four replicate wells and the error bars represent the standard deviation. A two-way ANOVA with Sidak’s multiple comparisons test was performed to evaluate statistical significant differences, ns: not significant, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.

    Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated from leukapheresis (pre-LEN timepoint) and whole blood (LEN timepoint) samples through lymphoprep gradient-centrifugation (Axis-Shield) following the manufacturer’s instructions before being cryopreserved.

    Techniques: Expressing, In Vitro, Enzyme-linked Immunospot, Standard Deviation

    Lenalidomide maintenance therapy results in an improved CD8 + T cell functionality, which is hampered by non-CD8 + T cells present in the periphery ( A ) PBMCs obtained at the pre LEN and the LEN timepoint were examined by IFN-γ ELISPOT to measure the CD8 + T-cell response to viral recall epitopes derived for CMV, EBV and influenza (CEF) ( n = 6). The number of spot forming units (SFU) as well as the activity in each well was determined. Bar graphs show the mean number of spots per million PBMCs or the mean activity of four replicate wells and the error bars show the standard deviation. Spot numbers and activity measured upon culture in the absence of peptide were subtracted. ( B – E ) CD8 + T cells purified from PBMC samples obtained at the pre LEN and LEN timepoint were examined by IFN-γ/IL-2/TNF-α FLUOROSPOT to characterize the CD8 + T-cell response to the CEF peptide pool. (B–C) Bar graphs show the mean number of spots per million CD8 + T cells of five replicate wells and the error bars show the standard deviation. Spot numbers and activity measured upon culture in the absence of peptide were subtracted. ( D ) Raw data showing three-color FLUOROSPOT results. One representative well is shown for each patient and timepoint. Cytokine-specific spots are shown in green (IFN-γ), red (TNF-α), blue (IL-2) or overlays of these colors. ( E ) The pie charts indicate the portion of mono- and polyfunctional CEF-specific CD8 + T cells. A two-way ANOVA with Sidak’s multiple comparisons test was performed to detect differences between the pre LEN and the LEN timepoint, ns: not significant, * p

    Journal: Oncotarget

    Article Title: Impact of lenalidomide maintenance on the immune environment of multiple myeloma patients with low tumor burden after autologous stem cell transplantation

    doi: 10.18632/oncotarget.24944

    Figure Lengend Snippet: Lenalidomide maintenance therapy results in an improved CD8 + T cell functionality, which is hampered by non-CD8 + T cells present in the periphery ( A ) PBMCs obtained at the pre LEN and the LEN timepoint were examined by IFN-γ ELISPOT to measure the CD8 + T-cell response to viral recall epitopes derived for CMV, EBV and influenza (CEF) ( n = 6). The number of spot forming units (SFU) as well as the activity in each well was determined. Bar graphs show the mean number of spots per million PBMCs or the mean activity of four replicate wells and the error bars show the standard deviation. Spot numbers and activity measured upon culture in the absence of peptide were subtracted. ( B – E ) CD8 + T cells purified from PBMC samples obtained at the pre LEN and LEN timepoint were examined by IFN-γ/IL-2/TNF-α FLUOROSPOT to characterize the CD8 + T-cell response to the CEF peptide pool. (B–C) Bar graphs show the mean number of spots per million CD8 + T cells of five replicate wells and the error bars show the standard deviation. Spot numbers and activity measured upon culture in the absence of peptide were subtracted. ( D ) Raw data showing three-color FLUOROSPOT results. One representative well is shown for each patient and timepoint. Cytokine-specific spots are shown in green (IFN-γ), red (TNF-α), blue (IL-2) or overlays of these colors. ( E ) The pie charts indicate the portion of mono- and polyfunctional CEF-specific CD8 + T cells. A two-way ANOVA with Sidak’s multiple comparisons test was performed to detect differences between the pre LEN and the LEN timepoint, ns: not significant, * p

    Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated from leukapheresis (pre-LEN timepoint) and whole blood (LEN timepoint) samples through lymphoprep gradient-centrifugation (Axis-Shield) following the manufacturer’s instructions before being cryopreserved.

    Techniques: Enzyme-linked Immunospot, Derivative Assay, Activity Assay, Standard Deviation, Purification

    Blockade of sialic-acid binding sites reveals BcR-dependent binding to influenza HA. A–B. Diffuse binding of rH1 to untreated human PBMC. Thawed PBMCs from an anonymous blood donor were first stained with Live/Dead then with an anti-CD20 mAb mixed with Alexa488-conjugated human serum albumin (PBS/HSA), or Alexa488-conjugated rH1 from the A/California/07/2009 strain (PBS/rH1), or a solution of sialopentasaccarides containing the Alexa488-conjugated rH1 (α-2,6-SPS/H1Cal). The strategy for gating on single live lymphocytes is shown in A; rH1 binding to CD20 negative (CD20 neg ) and CD20 + cells is shown in B. C. Blockade of sialic-acid binding sites reveals putative BcR-specifc binding of rH1. PBMCs from a single donor were split in 8 tubes (10 7 PBMC/each), 4 tubes were incubated with H3N2 mono-bulk subunit vaccine antigens from the A/Panama/2007/1999 strain (left dot plots), and 4 tubes with neuraminidase (right dot plots). Then the PBMCs were incubated with anti-CD20 and anti-CD27 mAbs and A488-HSA (upper plots), or A488-rH1 (lower plots). Shown is the distribution of rH1 binding on memory (CD27 + ) and naïve (CD27 neg ) B cells identified in the CD20 + gate. D. Specificity of rH1 binding. H1 + B-cells sorted from H3N2- (n = 1372) or neuraminidase- (Neur) (n = 2233) treated PBMCs were mixed with sorted autologous CD20 − cells in the ratio of 1∶200 and activated in vitro with CpG and IL-2 for 5 days. As controls, cultures of unsorted PBMCs, as well as of autologous CD20 neg cells mixed with H1-negative (H1 neg ) B cells sorted from neuraminidase-treated PBMCs (n = 30,000) in the ratio of 1∶10 were run for 5 days in the presence of CpG and IL-2. After 5 days, cells from each culture were harvested, washed, counted and assayed by ELISPOT to enumerate cells secreting IgG and H1N1-specific IgG. To facilitate comparisons between different cell cultures and across experiments depicted in different figures, the results are expressed as numbers of antibody secreting cells (ASC) normalized to 10 6 cultured cells assayed by ELISPOT.

    Journal: PLoS ONE

    Article Title: Ex Vivo Analysis of Human Memory B Lymphocytes Specific for A and B Influenza Hemagglutinin by Polychromatic Flow-Cytometry

    doi: 10.1371/journal.pone.0070620

    Figure Lengend Snippet: Blockade of sialic-acid binding sites reveals BcR-dependent binding to influenza HA. A–B. Diffuse binding of rH1 to untreated human PBMC. Thawed PBMCs from an anonymous blood donor were first stained with Live/Dead then with an anti-CD20 mAb mixed with Alexa488-conjugated human serum albumin (PBS/HSA), or Alexa488-conjugated rH1 from the A/California/07/2009 strain (PBS/rH1), or a solution of sialopentasaccarides containing the Alexa488-conjugated rH1 (α-2,6-SPS/H1Cal). The strategy for gating on single live lymphocytes is shown in A; rH1 binding to CD20 negative (CD20 neg ) and CD20 + cells is shown in B. C. Blockade of sialic-acid binding sites reveals putative BcR-specifc binding of rH1. PBMCs from a single donor were split in 8 tubes (10 7 PBMC/each), 4 tubes were incubated with H3N2 mono-bulk subunit vaccine antigens from the A/Panama/2007/1999 strain (left dot plots), and 4 tubes with neuraminidase (right dot plots). Then the PBMCs were incubated with anti-CD20 and anti-CD27 mAbs and A488-HSA (upper plots), or A488-rH1 (lower plots). Shown is the distribution of rH1 binding on memory (CD27 + ) and naïve (CD27 neg ) B cells identified in the CD20 + gate. D. Specificity of rH1 binding. H1 + B-cells sorted from H3N2- (n = 1372) or neuraminidase- (Neur) (n = 2233) treated PBMCs were mixed with sorted autologous CD20 − cells in the ratio of 1∶200 and activated in vitro with CpG and IL-2 for 5 days. As controls, cultures of unsorted PBMCs, as well as of autologous CD20 neg cells mixed with H1-negative (H1 neg ) B cells sorted from neuraminidase-treated PBMCs (n = 30,000) in the ratio of 1∶10 were run for 5 days in the presence of CpG and IL-2. After 5 days, cells from each culture were harvested, washed, counted and assayed by ELISPOT to enumerate cells secreting IgG and H1N1-specific IgG. To facilitate comparisons between different cell cultures and across experiments depicted in different figures, the results are expressed as numbers of antibody secreting cells (ASC) normalized to 10 6 cultured cells assayed by ELISPOT.

    Article Snippet: Samples were stained in tubes, each containing 107 PBMC, according to different protocols: i) For pre-saturation with mono-bulk vaccine antigens, PBMCs were first stained with Live/Dead Aqua (Invitrogen) diluted 1∶500 in 100 µl, for 20 min in the dark.

    Techniques: Binding Assay, Staining, Incubation, In Vitro, Enzyme-linked Immunospot, Cell Culture

    Vaccination induced changes in the pool of H1+ B-cells. PBMCs samples collected before (day 0) and at 3 and 6 weeks after vaccination from four seasonal influenza vaccinees, were pre-incubated with H3N2 subunit (from A/Panama/2007/1999) and then stained with rH1 A/Solomon Island/3/06) and mAbs anti-CD20, anti-CD27 and anti-human IgG. A. Dot plots gated on CD20 + B-cells showing the distribution of H1 + (middle panels) and H1 neg (bottom panels) B-cells from donor #a across: the mature memory (CD27 + ) and putatively naive (CD27 neg ) CD20+ B-cell subsets (upper panels); un-switched mature memory (CD27 + IgG neg ), IgG-switched mature (CD27 + IgG + ) and immature (CD27 neg IgG + ) memory B-cells. B. Numbers of circulating H1 + CD20 + B-cells in 4 vaccinees before and at 3 and 6 weeks after seasonal vaccination are overlaid with paired titers of antibodies inhibiting virus-induced hemmaglutination measured in their blood. The frequencies of H1 + B-cells are normalized according to the frequencies of CD20 + B-cells in 10 6 PBMCs. C. Distribution of circulating H1 + and H1 neg B-cells across same subsets identified in B in all vaccinees.

    Journal: PLoS ONE

    Article Title: Ex Vivo Analysis of Human Memory B Lymphocytes Specific for A and B Influenza Hemagglutinin by Polychromatic Flow-Cytometry

    doi: 10.1371/journal.pone.0070620

    Figure Lengend Snippet: Vaccination induced changes in the pool of H1+ B-cells. PBMCs samples collected before (day 0) and at 3 and 6 weeks after vaccination from four seasonal influenza vaccinees, were pre-incubated with H3N2 subunit (from A/Panama/2007/1999) and then stained with rH1 A/Solomon Island/3/06) and mAbs anti-CD20, anti-CD27 and anti-human IgG. A. Dot plots gated on CD20 + B-cells showing the distribution of H1 + (middle panels) and H1 neg (bottom panels) B-cells from donor #a across: the mature memory (CD27 + ) and putatively naive (CD27 neg ) CD20+ B-cell subsets (upper panels); un-switched mature memory (CD27 + IgG neg ), IgG-switched mature (CD27 + IgG + ) and immature (CD27 neg IgG + ) memory B-cells. B. Numbers of circulating H1 + CD20 + B-cells in 4 vaccinees before and at 3 and 6 weeks after seasonal vaccination are overlaid with paired titers of antibodies inhibiting virus-induced hemmaglutination measured in their blood. The frequencies of H1 + B-cells are normalized according to the frequencies of CD20 + B-cells in 10 6 PBMCs. C. Distribution of circulating H1 + and H1 neg B-cells across same subsets identified in B in all vaccinees.

    Article Snippet: Samples were stained in tubes, each containing 107 PBMC, according to different protocols: i) For pre-saturation with mono-bulk vaccine antigens, PBMCs were first stained with Live/Dead Aqua (Invitrogen) diluted 1∶500 in 100 µl, for 20 min in the dark.

    Techniques: Incubation, Staining

    Molecular cloning of HA + B lymphocytes. PBMCs from 4 anonymous blood bank donors were stained as in Figure 4B . Single H1 + , H3 + , or H1 neg H3 neg CD20 + B-cells were sorted to perform molecular cloning and analysis of their paired V H V L Ig regions as described in Material and Methods section. A–E. Distribution of V H (A), D H (B), J H (C), V k (D) and J k (E) gene use across arrays of B-cells sorted from each donor (16 and 18 HA + clones from donors #1 and #2; 35 HA + and 20 HA neg clones from donor 3; 16 HA + and 16 HA neg clones from donor #4. F–I. Number of mutations in H1 + and H1 neg CD20 + B-cells from donors #3 and #4, which cause dissimilar (F, H) or similar (G, I) amino acid substitutions in V H (G,I) and V L (F,H). NS and ** indicate not significant, or significant (p

    Journal: PLoS ONE

    Article Title: Ex Vivo Analysis of Human Memory B Lymphocytes Specific for A and B Influenza Hemagglutinin by Polychromatic Flow-Cytometry

    doi: 10.1371/journal.pone.0070620

    Figure Lengend Snippet: Molecular cloning of HA + B lymphocytes. PBMCs from 4 anonymous blood bank donors were stained as in Figure 4B . Single H1 + , H3 + , or H1 neg H3 neg CD20 + B-cells were sorted to perform molecular cloning and analysis of their paired V H V L Ig regions as described in Material and Methods section. A–E. Distribution of V H (A), D H (B), J H (C), V k (D) and J k (E) gene use across arrays of B-cells sorted from each donor (16 and 18 HA + clones from donors #1 and #2; 35 HA + and 20 HA neg clones from donor 3; 16 HA + and 16 HA neg clones from donor #4. F–I. Number of mutations in H1 + and H1 neg CD20 + B-cells from donors #3 and #4, which cause dissimilar (F, H) or similar (G, I) amino acid substitutions in V H (G,I) and V L (F,H). NS and ** indicate not significant, or significant (p

    Article Snippet: Samples were stained in tubes, each containing 107 PBMC, according to different protocols: i) For pre-saturation with mono-bulk vaccine antigens, PBMCs were first stained with Live/Dead Aqua (Invitrogen) diluted 1∶500 in 100 µl, for 20 min in the dark.

    Techniques: Molecular Cloning, Staining, Clone Assay

    Identification of B lymphocytes specific for HA from A and B influenza strains in ex vivo PBMCs samples. PBMCs from different anonymous blood donors were pre-incubated with vaccine mono-bulk subunits from the B/Brisbane/60/2008 (B/HA pretreatment), or the H3N2 A/Panama/2007/1999 strain (A/HA pretreatment) and then stained HSA, rH3 (from A/Brisbane/10/2007), rH1 (from A/California/07/2009), or B/HA (from B/Brisbane/60/2008), as indicated. A. Staining pattern observed on CD20 + cells in PBMCs stained with the different rHA bait. The rectangular gates identify brilliant HA+ B-cells; the dotted vertical lines mark the gates used to sort HA + B-cells for the ELISPOT assays. B. Expression of the CD27 memory marker on HA + and HA neg B cells identified based on the sorting gates. C. H3 + (n = 15,234), H1 + (n = 6482) and B/HA + (n = 26,803) B-cells identified in A were sorted, mixed with autologous CD20 neg cells (in the ratio of 1∶20, 1∶100 and 1∶33) and activated with CpG and IL-2 for 5 days in vitro . Unsorted PBMCs and CD20 neg cells mixed with HA neg B cells were also cultured in the same manner, as controls. After 5 days cultured cells were harvested and assayed by ELISPOT for the number of cells secreting IgG and IgG specific for mono-bulk subunits from the vaccine strain homologous to the sorting bait. Results are expressed as numbers of antibody secreting cells (ASC) normalized to 10 6 cultured cells assayed by ELISPOT. Nd indicates undetectable ASC.

    Journal: PLoS ONE

    Article Title: Ex Vivo Analysis of Human Memory B Lymphocytes Specific for A and B Influenza Hemagglutinin by Polychromatic Flow-Cytometry

    doi: 10.1371/journal.pone.0070620

    Figure Lengend Snippet: Identification of B lymphocytes specific for HA from A and B influenza strains in ex vivo PBMCs samples. PBMCs from different anonymous blood donors were pre-incubated with vaccine mono-bulk subunits from the B/Brisbane/60/2008 (B/HA pretreatment), or the H3N2 A/Panama/2007/1999 strain (A/HA pretreatment) and then stained HSA, rH3 (from A/Brisbane/10/2007), rH1 (from A/California/07/2009), or B/HA (from B/Brisbane/60/2008), as indicated. A. Staining pattern observed on CD20 + cells in PBMCs stained with the different rHA bait. The rectangular gates identify brilliant HA+ B-cells; the dotted vertical lines mark the gates used to sort HA + B-cells for the ELISPOT assays. B. Expression of the CD27 memory marker on HA + and HA neg B cells identified based on the sorting gates. C. H3 + (n = 15,234), H1 + (n = 6482) and B/HA + (n = 26,803) B-cells identified in A were sorted, mixed with autologous CD20 neg cells (in the ratio of 1∶20, 1∶100 and 1∶33) and activated with CpG and IL-2 for 5 days in vitro . Unsorted PBMCs and CD20 neg cells mixed with HA neg B cells were also cultured in the same manner, as controls. After 5 days cultured cells were harvested and assayed by ELISPOT for the number of cells secreting IgG and IgG specific for mono-bulk subunits from the vaccine strain homologous to the sorting bait. Results are expressed as numbers of antibody secreting cells (ASC) normalized to 10 6 cultured cells assayed by ELISPOT. Nd indicates undetectable ASC.

    Article Snippet: Samples were stained in tubes, each containing 107 PBMC, according to different protocols: i) For pre-saturation with mono-bulk vaccine antigens, PBMCs were first stained with Live/Dead Aqua (Invitrogen) diluted 1∶500 in 100 µl, for 20 min in the dark.

    Techniques: Ex Vivo, Incubation, Staining, Enzyme-linked Immunospot, Expressing, Marker, In Vitro, Cell Culture

    H1 + IgG + MBCs frequencies measured by flow-cytometry and by ELISPOT correlated linearly. A–B. Specificity of the staining with the rH1 bait from A/Solomon Island/3/06. PBMCs (1.6×10 8 ) from anonymous blood donors were stained with Live/Dead, incubated with an H3N2 mono-bulk vaccine subunit (from A/Panama/2007/1999), and then stained with Alexa647-conjugated HSA (6×10 7 ), or Alexa647-conjugated rH1 (1×10 8 ), and with an antiCD20 mAb. A. Binding pattern of HSA (A647-HSA; left panel) and of rH1 (A647-rH1; right panel) in the CD20 + B-cell gates. B. H1 + B-cells identified in A were sorted (n = 8215), mixed with autologous CD20 neg cells in the ratio of 1∶50 and activated with CpG and IL-2 for 5 days in vitro . Unsorted PBMC and CD20 neg cells were also cultured in the same manner as controls. After 5 days, equal numbers of cultured cells were harvested and assayed by ELISPOT for numbers of cells secreting IgG and IgG specific for H1N1 (from A/Solomon Island/3/06). Results are expressed as number of antibody secreting cells (ASC) normalized to 10 6 cultured cells assayed by ELISPOT. Nd indicates undetectable ASC. C. Distribution of IgG + B-cells among H1 neg and H1 + B cells expressing or not the CD27 B cell memory marker; shown is one representative subject. D. Replicates of frozen PBMCs from 4 anonymous blood donors were assayed by conventional ELISPOT, or incubated with an H3N2 mono-bulk vaccine subunit and stained with rH1, and anti-CD20 plus anti-human IgG antibodies. The scatter plot depicts paired values of H1 + IgG + B-cell frequencies measured by flow-cytometry (y-axis) and by ELISPOT (x-axis) across three different experimental sessions. Shown are: the regression line with the related 95% confidence interval (gray areas), slope, intercepts, R 2 and p-value. E. Variability plot showing mean standard deviations of the measurements done by ELISPOT and flow-cytometry. The three dotted lines mark the grand mean and the upper and lower control limits.

    Journal: PLoS ONE

    Article Title: Ex Vivo Analysis of Human Memory B Lymphocytes Specific for A and B Influenza Hemagglutinin by Polychromatic Flow-Cytometry

    doi: 10.1371/journal.pone.0070620

    Figure Lengend Snippet: H1 + IgG + MBCs frequencies measured by flow-cytometry and by ELISPOT correlated linearly. A–B. Specificity of the staining with the rH1 bait from A/Solomon Island/3/06. PBMCs (1.6×10 8 ) from anonymous blood donors were stained with Live/Dead, incubated with an H3N2 mono-bulk vaccine subunit (from A/Panama/2007/1999), and then stained with Alexa647-conjugated HSA (6×10 7 ), or Alexa647-conjugated rH1 (1×10 8 ), and with an antiCD20 mAb. A. Binding pattern of HSA (A647-HSA; left panel) and of rH1 (A647-rH1; right panel) in the CD20 + B-cell gates. B. H1 + B-cells identified in A were sorted (n = 8215), mixed with autologous CD20 neg cells in the ratio of 1∶50 and activated with CpG and IL-2 for 5 days in vitro . Unsorted PBMC and CD20 neg cells were also cultured in the same manner as controls. After 5 days, equal numbers of cultured cells were harvested and assayed by ELISPOT for numbers of cells secreting IgG and IgG specific for H1N1 (from A/Solomon Island/3/06). Results are expressed as number of antibody secreting cells (ASC) normalized to 10 6 cultured cells assayed by ELISPOT. Nd indicates undetectable ASC. C. Distribution of IgG + B-cells among H1 neg and H1 + B cells expressing or not the CD27 B cell memory marker; shown is one representative subject. D. Replicates of frozen PBMCs from 4 anonymous blood donors were assayed by conventional ELISPOT, or incubated with an H3N2 mono-bulk vaccine subunit and stained with rH1, and anti-CD20 plus anti-human IgG antibodies. The scatter plot depicts paired values of H1 + IgG + B-cell frequencies measured by flow-cytometry (y-axis) and by ELISPOT (x-axis) across three different experimental sessions. Shown are: the regression line with the related 95% confidence interval (gray areas), slope, intercepts, R 2 and p-value. E. Variability plot showing mean standard deviations of the measurements done by ELISPOT and flow-cytometry. The three dotted lines mark the grand mean and the upper and lower control limits.

    Article Snippet: Samples were stained in tubes, each containing 107 PBMC, according to different protocols: i) For pre-saturation with mono-bulk vaccine antigens, PBMCs were first stained with Live/Dead Aqua (Invitrogen) diluted 1∶500 in 100 µl, for 20 min in the dark.

    Techniques: Flow Cytometry, Cytometry, Enzyme-linked Immunospot, Staining, Incubation, Binding Assay, In Vitro, Cell Culture, Expressing, Marker

    Simultaneous identification of B lymphocytes specific for HA from different influenza strains in ex vivo PBMCs samples. A. PBMCs from an anonymous blood donor were pre-incubated with subunits from either B/Brisbane/60/2008 or A/Panama/2007/1999 (H3N2), and then stained with anti-CD20 mAb, HSA conjugated with A488 and A647, with A647-rH3 (from A/Brisbane/10/2007) and A488-rH1 (from A/California/07/09), or with A647-rB/HA (from B/Brisbane/60/2008) and A488-rH1 (from A/California/07/09). The staining patterns observed in the CD20 + B-cell gate are shown. B. PBMCs from 16 anonymous blood donors were pre-saturated with B/Brisbane/60/2008 and then stained with anti-CD20 mAb, A647-rH3 (from A/Brisbane/10/2007) and A488-rH1 (from A/California/07/09). The scatter plot depicts paired values of H1 + (y-axis) and H3 + (x-axis) B-cells. The insert box plot depicts the distribution of H1 + , H3 + and H1 + H3 + B-cells in the same 16 donors. Mean values are indicated by dotted lines.

    Journal: PLoS ONE

    Article Title: Ex Vivo Analysis of Human Memory B Lymphocytes Specific for A and B Influenza Hemagglutinin by Polychromatic Flow-Cytometry

    doi: 10.1371/journal.pone.0070620

    Figure Lengend Snippet: Simultaneous identification of B lymphocytes specific for HA from different influenza strains in ex vivo PBMCs samples. A. PBMCs from an anonymous blood donor were pre-incubated with subunits from either B/Brisbane/60/2008 or A/Panama/2007/1999 (H3N2), and then stained with anti-CD20 mAb, HSA conjugated with A488 and A647, with A647-rH3 (from A/Brisbane/10/2007) and A488-rH1 (from A/California/07/09), or with A647-rB/HA (from B/Brisbane/60/2008) and A488-rH1 (from A/California/07/09). The staining patterns observed in the CD20 + B-cell gate are shown. B. PBMCs from 16 anonymous blood donors were pre-saturated with B/Brisbane/60/2008 and then stained with anti-CD20 mAb, A647-rH3 (from A/Brisbane/10/2007) and A488-rH1 (from A/California/07/09). The scatter plot depicts paired values of H1 + (y-axis) and H3 + (x-axis) B-cells. The insert box plot depicts the distribution of H1 + , H3 + and H1 + H3 + B-cells in the same 16 donors. Mean values are indicated by dotted lines.

    Article Snippet: Samples were stained in tubes, each containing 107 PBMC, according to different protocols: i) For pre-saturation with mono-bulk vaccine antigens, PBMCs were first stained with Live/Dead Aqua (Invitrogen) diluted 1∶500 in 100 µl, for 20 min in the dark.

    Techniques: Ex Vivo, Incubation, Staining

    Measuring CTLA-4 and Fas expression by CD4 + ,FOXP3 + T cells as well as apoptosis of CD4 + ,CD25 bright and CD25 − T cells in young and elderly humans. (A – D) PBMCs from young (n = 15) and elderly (n = 15) individuals were stained with Abs

    Journal: Mechanisms of ageing and development

    Article Title: Aging and human CD4+ regulatory T cells

    doi: 10.1016/j.mad.2009.06.003

    Figure Lengend Snippet: Measuring CTLA-4 and Fas expression by CD4 + ,FOXP3 + T cells as well as apoptosis of CD4 + ,CD25 bright and CD25 − T cells in young and elderly humans. (A – D) PBMCs from young (n = 15) and elderly (n = 15) individuals were stained with Abs

    Article Snippet: Peripheral blood mononuclear cells (PBMCs) were stained with mouse anti-human CD4 and CD25 antibodies (Abs) (BD Pharmingen, San Jose, CA) followed by washing, permeabilization with permeabilizing buffer and staining with mouse anti-FOXP3 Abs (clone PCH101, eBioscience, San Diego, CA).

    Techniques: Expressing, Staining

    Correlation of CD25, FOXP3 and IL-7Rα expression by CD4 + T cells in young and elderly humans. Purified peripheral blood mononuclear cells (PBMCs) from young (n = 10) and elderly (n = 10) individuals were stained with Abs to CD4 and CD25, followed

    Journal: Mechanisms of ageing and development

    Article Title: Aging and human CD4+ regulatory T cells

    doi: 10.1016/j.mad.2009.06.003

    Figure Lengend Snippet: Correlation of CD25, FOXP3 and IL-7Rα expression by CD4 + T cells in young and elderly humans. Purified peripheral blood mononuclear cells (PBMCs) from young (n = 10) and elderly (n = 10) individuals were stained with Abs to CD4 and CD25, followed

    Article Snippet: Peripheral blood mononuclear cells (PBMCs) were stained with mouse anti-human CD4 and CD25 antibodies (Abs) (BD Pharmingen, San Jose, CA) followed by washing, permeabilization with permeabilizing buffer and staining with mouse anti-FOXP3 Abs (clone PCH101, eBioscience, San Diego, CA).

    Techniques: Expressing, Purification, Staining

    Comparing the frequency of CD4 + ,FOXP3 + T cells and their naïve (CD45RA + ) and memory (CD45RA − ) phenotypes between young and elderly humans. PBMCs from young (n = 15) and elderly (n = 15) individuals were stained with Abs to CD4, CD45RA

    Journal: Mechanisms of ageing and development

    Article Title: Aging and human CD4+ regulatory T cells

    doi: 10.1016/j.mad.2009.06.003

    Figure Lengend Snippet: Comparing the frequency of CD4 + ,FOXP3 + T cells and their naïve (CD45RA + ) and memory (CD45RA − ) phenotypes between young and elderly humans. PBMCs from young (n = 15) and elderly (n = 15) individuals were stained with Abs to CD4, CD45RA

    Article Snippet: Peripheral blood mononuclear cells (PBMCs) were stained with mouse anti-human CD4 and CD25 antibodies (Abs) (BD Pharmingen, San Jose, CA) followed by washing, permeabilization with permeabilizing buffer and staining with mouse anti-FOXP3 Abs (clone PCH101, eBioscience, San Diego, CA).

    Techniques: Staining

    Comparing the capacity of CD4 + ,CD25 bright T cells in suppressing the proliferation and cytokine production of CD4 + ,CD25 − T cells between young and elderly humans. PBMCs from young and elderly individuals were stained with Abs to CD4 and CD25 and

    Journal: Mechanisms of ageing and development

    Article Title: Aging and human CD4+ regulatory T cells

    doi: 10.1016/j.mad.2009.06.003

    Figure Lengend Snippet: Comparing the capacity of CD4 + ,CD25 bright T cells in suppressing the proliferation and cytokine production of CD4 + ,CD25 − T cells between young and elderly humans. PBMCs from young and elderly individuals were stained with Abs to CD4 and CD25 and

    Article Snippet: Peripheral blood mononuclear cells (PBMCs) were stained with mouse anti-human CD4 and CD25 antibodies (Abs) (BD Pharmingen, San Jose, CA) followed by washing, permeabilization with permeabilizing buffer and staining with mouse anti-FOXP3 Abs (clone PCH101, eBioscience, San Diego, CA).

    Techniques: Staining

    Chemokine receptor expression on CD4 + ,FOXP3 + T cells in young and elderly humans. PBMCs from young (n = 15) and elderly (n = 15) individuals were stained with Abs to CD4, CCR7, CCR4, CCR5, CXCR3 or isotype Abs, followed by permeabilization and staining

    Journal: Mechanisms of ageing and development

    Article Title: Aging and human CD4+ regulatory T cells

    doi: 10.1016/j.mad.2009.06.003

    Figure Lengend Snippet: Chemokine receptor expression on CD4 + ,FOXP3 + T cells in young and elderly humans. PBMCs from young (n = 15) and elderly (n = 15) individuals were stained with Abs to CD4, CCR7, CCR4, CCR5, CXCR3 or isotype Abs, followed by permeabilization and staining

    Article Snippet: Peripheral blood mononuclear cells (PBMCs) were stained with mouse anti-human CD4 and CD25 antibodies (Abs) (BD Pharmingen, San Jose, CA) followed by washing, permeabilization with permeabilizing buffer and staining with mouse anti-FOXP3 Abs (clone PCH101, eBioscience, San Diego, CA).

    Techniques: Expressing, Staining

    Signal scatter plots comparing the intensities of gene expressions on microarray chips within Ficoll and CPT-derived PBMC and their immune cell subsets for each healthy donor tested. The expression signals of Ficoll-derived cells were plotted against the expression signals of CPT-derived cells for each cell type and donor. A line of perfect correlation is indicated in all plots. Pearson’s correlation coefficients between each pair of samples are shown at the bottom right of each plot. The data showed that the pattern of gene expression was very similar between Ficoll and CPT-derived cell types, as indicated by the high correlation coefficients

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Signal scatter plots comparing the intensities of gene expressions on microarray chips within Ficoll and CPT-derived PBMC and their immune cell subsets for each healthy donor tested. The expression signals of Ficoll-derived cells were plotted against the expression signals of CPT-derived cells for each cell type and donor. A line of perfect correlation is indicated in all plots. Pearson’s correlation coefficients between each pair of samples are shown at the bottom right of each plot. The data showed that the pattern of gene expression was very similar between Ficoll and CPT-derived cell types, as indicated by the high correlation coefficients

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Microarray, Cycling Probe Technology, Derivative Assay, Expressing

    Yield and viability of immune cell subsets prepared from PBMC isolated by either Ficoll or CPT protocol. a The mean number of CD19+, CD8+, CD14+, and CD4+ cells positively selected from Ficoll- and CPT-isolated PBMC collected from 6 healthy donors. The horizontal line denotes the means and each symbol represents an individual cell type isolated from Ficoll-PBMC (filled symbols) or CPT-PBMC (empty symbols) from each of 6 donors. b The mean viability of the same cell subsets. No significant differences ( P

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Yield and viability of immune cell subsets prepared from PBMC isolated by either Ficoll or CPT protocol. a The mean number of CD19+, CD8+, CD14+, and CD4+ cells positively selected from Ficoll- and CPT-isolated PBMC collected from 6 healthy donors. The horizontal line denotes the means and each symbol represents an individual cell type isolated from Ficoll-PBMC (filled symbols) or CPT-PBMC (empty symbols) from each of 6 donors. b The mean viability of the same cell subsets. No significant differences ( P

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Isolation, Cycling Probe Technology

    Principle component analysis of gene expression showing distinct clusters between individual immune cell subsets but not between the same subsets derived from PBMC isolated by CPT or Ficoll protocols. Principle component analysis (PCA) was performed based on expression of all genes from the microarray. Individual immune cell types are represented by different colors: CD19+ B cells, blue; CD8+ T cells, green; CD14+ monocytes, purple; CD4+ T cells, orange; and total PBMC, red. The method used for PBMC isolation is represented by different symbols of differing size: CPT-based procedure by large circles and Ficoll-based procedure by small circles. PC#1, principle component #1; PC#2, principle component #2

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Principle component analysis of gene expression showing distinct clusters between individual immune cell subsets but not between the same subsets derived from PBMC isolated by CPT or Ficoll protocols. Principle component analysis (PCA) was performed based on expression of all genes from the microarray. Individual immune cell types are represented by different colors: CD19+ B cells, blue; CD8+ T cells, green; CD14+ monocytes, purple; CD4+ T cells, orange; and total PBMC, red. The method used for PBMC isolation is represented by different symbols of differing size: CPT-based procedure by large circles and Ficoll-based procedure by small circles. PC#1, principle component #1; PC#2, principle component #2

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Expressing, Derivative Assay, Isolation, Cycling Probe Technology, Microarray

    Yield and quality of RNA recovered from total PBMC and their individual immune cell subsets did not significantly differ between PBMC isolated by Ficoll and CPT methods. a The mean amount of RNA per cell and ( b ) the mean RIN of RNA preparations extracted from total PBMC and their individual immune cell subsets derived from PBMC prepared by Ficoll and CPT methods from 6 health donors. There were no significant differences ( P

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Yield and quality of RNA recovered from total PBMC and their individual immune cell subsets did not significantly differ between PBMC isolated by Ficoll and CPT methods. a The mean amount of RNA per cell and ( b ) the mean RIN of RNA preparations extracted from total PBMC and their individual immune cell subsets derived from PBMC prepared by Ficoll and CPT methods from 6 health donors. There were no significant differences ( P

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Isolation, Cycling Probe Technology, Derivative Assay

    Similar quantities of DNA with comparable quality were obtained from PBMC and immune cell subsets following either Ficoll or CPT protocol of PBMC isolation. a The mean amount of DNA extracted per cell and ( b ) the mean of the 260/280 ratios, as measure of DNA quality, were compared between DNA preparations obtained from total PBMC and immune cell subsets prepared from PBMC isolated by Ficoll and CPT methods from 6 healthy donors. There were no significant differences ( P

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Similar quantities of DNA with comparable quality were obtained from PBMC and immune cell subsets following either Ficoll or CPT protocol of PBMC isolation. a The mean amount of DNA extracted per cell and ( b ) the mean of the 260/280 ratios, as measure of DNA quality, were compared between DNA preparations obtained from total PBMC and immune cell subsets prepared from PBMC isolated by Ficoll and CPT methods from 6 healthy donors. There were no significant differences ( P

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Cycling Probe Technology, Isolation

    Schematic outline of the study. PBMC from 6 healthy donors were isolated using Ficoll-Paque gradient fractionation or BD Vacutainer CPT protocol, and cell yield and purity were compared. Subsequently, immune cell subsets were separated by positive selection from PBMC obtained by both methods, and yields and viabilities of the subsets were determined and compared. RNA and DNA were extracted from total PBMC isolated by both protocols and from their subsets, and the nucleic acid yield and quality compared. Finally, gene expression analysis of individual immune cell subsets and total PBMC obtained by Ficoll and CPT isolation methods were performed and the results compared. LN 2 , liquid nitrogen

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Schematic outline of the study. PBMC from 6 healthy donors were isolated using Ficoll-Paque gradient fractionation or BD Vacutainer CPT protocol, and cell yield and purity were compared. Subsequently, immune cell subsets were separated by positive selection from PBMC obtained by both methods, and yields and viabilities of the subsets were determined and compared. RNA and DNA were extracted from total PBMC isolated by both protocols and from their subsets, and the nucleic acid yield and quality compared. Finally, gene expression analysis of individual immune cell subsets and total PBMC obtained by Ficoll and CPT isolation methods were performed and the results compared. LN 2 , liquid nitrogen

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Isolation, Fractionation, Cycling Probe Technology, Selection, Expressing

    Yield and viability of PBMC isolated by the Ficoll and CPT protocols. a The mean numbers of PBMC per ml of blood obtained by Ficoll or CPT isolation procedure from 6 healthy donors. b The mean viability of PBMC freshly isolated from the same 6 healthy donors by either Ficoll gradient separation or CPT technique. No significant differences ( P

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Yield and viability of PBMC isolated by the Ficoll and CPT protocols. a The mean numbers of PBMC per ml of blood obtained by Ficoll or CPT isolation procedure from 6 healthy donors. b The mean viability of PBMC freshly isolated from the same 6 healthy donors by either Ficoll gradient separation or CPT technique. No significant differences ( P

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Isolation, Cycling Probe Technology

    Purity of immune cell subsets obtained from PBMC isolated by either Ficoll or CPT procedures. CD19+, CD8+, CD14+, and CD4+ cell subsets were sequentially separated from PBMC isolated by Ficoll and CPT protocols from the same donor and their purity was determined by flow cytometry using antibodies against surface markers specific for individual immune cell types. Filled histograms were given by appropriate immunoglobulin isotype controls. Gates for determining positivity were established using isotype controls so that ~99.0 % of events were negative

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Purity of immune cell subsets obtained from PBMC isolated by either Ficoll or CPT procedures. CD19+, CD8+, CD14+, and CD4+ cell subsets were sequentially separated from PBMC isolated by Ficoll and CPT protocols from the same donor and their purity was determined by flow cytometry using antibodies against surface markers specific for individual immune cell types. Filled histograms were given by appropriate immunoglobulin isotype controls. Gates for determining positivity were established using isotype controls so that ~99.0 % of events were negative

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Isolation, Cycling Probe Technology, Flow Cytometry, Cytometry

    Post-cryopreservation recovery and viability of PBMC isolated by the Ficoll and CPT methods. a The mean percent recovery after cryopreservation of PBMC collected using the Ficoll and CPT protocols. Percent recovery was calculated by dividing the number of PBMC recovered after thawing by the number of cells that were cryopreserved. b The mean viability of recovered PBMC isolated by the Ficoll and CPT technique. No significant differences ( P

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Post-cryopreservation recovery and viability of PBMC isolated by the Ficoll and CPT methods. a The mean percent recovery after cryopreservation of PBMC collected using the Ficoll and CPT protocols. Percent recovery was calculated by dividing the number of PBMC recovered after thawing by the number of cells that were cryopreserved. b The mean viability of recovered PBMC isolated by the Ficoll and CPT technique. No significant differences ( P

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Isolation, Cycling Probe Technology

    CD69 expression in response to CMV antigens and to the influenza vaccine. PBMCs from elderly individuals were stimulated for 18 h and CD69 expression was evaluated by flow cytometry. a Representative dot plots of the frequency of CMV- and influenza vaccine-specific CD4 T cells with and without NKG2D expression. Percentages of positive cells in this representative experiment are expressed in dot plots. b Histograms summarize the percentage of CD69-cells positive for CMV and influenza vaccine in the CD4+ CD28 null NKG2D+ ( black bars ) and CD4+ CD28 null NKG2D- subsets ( white bars ) obtained from 11 elderly donors. Paired t test was used to compare paired frequencies

    Journal: Age

    Article Title: NKG2D expression in CD4+ T lymphocytes as a marker of senescence in the aged immune system

    doi: 10.1007/s11357-010-9200-6

    Figure Lengend Snippet: CD69 expression in response to CMV antigens and to the influenza vaccine. PBMCs from elderly individuals were stimulated for 18 h and CD69 expression was evaluated by flow cytometry. a Representative dot plots of the frequency of CMV- and influenza vaccine-specific CD4 T cells with and without NKG2D expression. Percentages of positive cells in this representative experiment are expressed in dot plots. b Histograms summarize the percentage of CD69-cells positive for CMV and influenza vaccine in the CD4+ CD28 null NKG2D+ ( black bars ) and CD4+ CD28 null NKG2D- subsets ( white bars ) obtained from 11 elderly donors. Paired t test was used to compare paired frequencies

    Article Snippet: To analyze the differentiation status of CD4+ T cells, peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on Ficoll–Hypaque gradients (Lymphoprep; Nycomed, Oslo, Norway).

    Techniques: Expressing, Flow Cytometry, Cytometry

    Differentiated status of CD4+ subsets defined by IL-2/IFN-γ production and TREC content. a PBMCs were stimulated for 6 h with soluble anti-CD3 (1 μg/mL). The responder cells were analyzed for intracellular IL-2-APC staining in the indicated populations. b Cells were treated as described previously and IFN-γ expression was analyzed by intracellular staining. Percentages of positive cells in the indicated populations in this representative experiment are expressed on the histogram-plots. Histograms summarize the percentage of IFN-γ-positive cells in CD4+ CD28 null and in CD4+ CD28+ lymphocytes NKG2D− and NKG2D + (mean ± SEM) from the six elderly donors tested. A paired t test was used to compare paired means. c Dose-response curves of the IFN-γ production by CD4 subsets defined by CD28 and NKG2D expression in response to anti-CD3. The cells were cultured for 6 h in medium alone or with increasing concentrations of anti-CD3 (1–1,000 ng/mL). A fluorescence analysis was carried out as previously described. One representative experiment of three is shown. d Quantification of TREC copy number. CD28+, CD28 null NKG2D − , and CD28 null NKG2D+ populations were isolated by sorting (magnetic bead separation) and the TREC copy number was determined by real-time PCR. Experiments were conducted in duplicate and bars represented results from three aging donors (mean ± SEM)

    Journal: Age

    Article Title: NKG2D expression in CD4+ T lymphocytes as a marker of senescence in the aged immune system

    doi: 10.1007/s11357-010-9200-6

    Figure Lengend Snippet: Differentiated status of CD4+ subsets defined by IL-2/IFN-γ production and TREC content. a PBMCs were stimulated for 6 h with soluble anti-CD3 (1 μg/mL). The responder cells were analyzed for intracellular IL-2-APC staining in the indicated populations. b Cells were treated as described previously and IFN-γ expression was analyzed by intracellular staining. Percentages of positive cells in the indicated populations in this representative experiment are expressed on the histogram-plots. Histograms summarize the percentage of IFN-γ-positive cells in CD4+ CD28 null and in CD4+ CD28+ lymphocytes NKG2D− and NKG2D + (mean ± SEM) from the six elderly donors tested. A paired t test was used to compare paired means. c Dose-response curves of the IFN-γ production by CD4 subsets defined by CD28 and NKG2D expression in response to anti-CD3. The cells were cultured for 6 h in medium alone or with increasing concentrations of anti-CD3 (1–1,000 ng/mL). A fluorescence analysis was carried out as previously described. One representative experiment of three is shown. d Quantification of TREC copy number. CD28+, CD28 null NKG2D − , and CD28 null NKG2D+ populations were isolated by sorting (magnetic bead separation) and the TREC copy number was determined by real-time PCR. Experiments were conducted in duplicate and bars represented results from three aging donors (mean ± SEM)

    Article Snippet: To analyze the differentiation status of CD4+ T cells, peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on Ficoll–Hypaque gradients (Lymphoprep; Nycomed, Oslo, Norway).

    Techniques: Staining, Expressing, Cell Culture, Fluorescence, Isolation, Real-time Polymerase Chain Reaction

    Single cell profiling identifies CD96 high and CD96 low subsets of Th9 cells. ( A – D ) Single cell gene expression analysis of CD44 + alloreactive T cells sorted from nonpolarized (w/o; n = 100), Th1 ( n = 67), and Th9 ( n = 113) polarized BALB/c cocultures. See Table S2 for a list of all genes measured. ( A ) Principal component analysis of gene expression in indicated Th subsets. ( B ) Nonsupervised hierarchical clustering analysis (Pearson’s correlation) comparing gene expression in Th subsets. Analysis includes all genes with detectable expression. ( C ) Box plots of most differentially expressed genes. Bonferroni-corrected ANOVA P values: Il9 ( P = 1.4 × 10 −126 ), Il2ra ( P = 1.4 × 10 −65 ), Cd83 ( P = 7.1 × 10 −64 ), Il4ra ( P = 1.2 × 10 −52 ), Gata3 ( P = 3.6 × 10 −47 ), Il1r1 ( P = 5.5 × 10 −47 ), Ccr4 ( P = 2.2 × 10 −46 ), and Cd28 ( P = 2.3 × 10 −43 ). ( D ) Nonsupervised hierarchical clustering analysis (complete linkage method) of gene expression in CD44 + T cells sorted from alloreactive BALB/c Th9 cultures. Displayed genes contributed to clustering. Frame indicates a cluster of Th9 cells characterized by high Cd96 expression. ( E ) Contour plots of surface CD96 expression in CD44 + polarized alloreactive BALB/c T cells, including fluorescence minus one (FMO) staining control (Ctrl). ( F ) Comparison of CD96 expression levels in IL-9 + and IFN-γ + human CD4 + T cells. PBMCs from blood of 10 healthy individuals were stimulated ex vivo with Staphylococcus enterotoxin B (SEB) for 24 h. A one-tailed Wilcoxon matched-pairs signed rank test was applied. gMFI, geometric mean fluorescence intensity.

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

    Article Title: CD96 expression determines the inflammatory potential of IL-9–producing Th9 cells

    doi: 10.1073/pnas.1708329115

    Figure Lengend Snippet: Single cell profiling identifies CD96 high and CD96 low subsets of Th9 cells. ( A – D ) Single cell gene expression analysis of CD44 + alloreactive T cells sorted from nonpolarized (w/o; n = 100), Th1 ( n = 67), and Th9 ( n = 113) polarized BALB/c cocultures. See Table S2 for a list of all genes measured. ( A ) Principal component analysis of gene expression in indicated Th subsets. ( B ) Nonsupervised hierarchical clustering analysis (Pearson’s correlation) comparing gene expression in Th subsets. Analysis includes all genes with detectable expression. ( C ) Box plots of most differentially expressed genes. Bonferroni-corrected ANOVA P values: Il9 ( P = 1.4 × 10 −126 ), Il2ra ( P = 1.4 × 10 −65 ), Cd83 ( P = 7.1 × 10 −64 ), Il4ra ( P = 1.2 × 10 −52 ), Gata3 ( P = 3.6 × 10 −47 ), Il1r1 ( P = 5.5 × 10 −47 ), Ccr4 ( P = 2.2 × 10 −46 ), and Cd28 ( P = 2.3 × 10 −43 ). ( D ) Nonsupervised hierarchical clustering analysis (complete linkage method) of gene expression in CD44 + T cells sorted from alloreactive BALB/c Th9 cultures. Displayed genes contributed to clustering. Frame indicates a cluster of Th9 cells characterized by high Cd96 expression. ( E ) Contour plots of surface CD96 expression in CD44 + polarized alloreactive BALB/c T cells, including fluorescence minus one (FMO) staining control (Ctrl). ( F ) Comparison of CD96 expression levels in IL-9 + and IFN-γ + human CD4 + T cells. PBMCs from blood of 10 healthy individuals were stimulated ex vivo with Staphylococcus enterotoxin B (SEB) for 24 h. A one-tailed Wilcoxon matched-pairs signed rank test was applied. gMFI, geometric mean fluorescence intensity.

    Article Snippet: For quantification of intracellular cytokines in human CD4+ T cells, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Biochrom) gradient centrifugation and restimulated with 1 µg/mL staphylococcal enterotoxin B (SEB) (Sigma-Aldrich) for 24 h at 37 °C.

    Techniques: Expressing, Fluorescence, Staining, Ex Vivo, One-tailed Test

    Semi-quantitative PCR analysis of BLV pol detects differences in proviral load between BLV SGV and BLV-rabbits. PBMC were harvested at month indicated after challenge with 1 × 10 8 FLK/BLV cells from indicated rabbits that had been infected with BLV SGV for 24 months or from age-matched naïve controls. DNA from 50,000 PMBC was subjected to nested PCR and gel electrophoresis and stained with ethidium bromide. The position of the 591-bp amplicon is designated by the labeled arrow. In parallel, DNA from 20,000 PBMC was subjected to PCR with primers complementary to β- actin . The position of the 594-bp amplicon is designated by the labeled arrow and the signals were quantified by densitometry.

    Journal: Virology

    Article Title: Long-term infection with retroviral structural gene vector provides protection against bovine leukemia virus disease in rabbits

    doi: 10.1016/j.virol.2004.09.001

    Figure Lengend Snippet: Semi-quantitative PCR analysis of BLV pol detects differences in proviral load between BLV SGV and BLV-rabbits. PBMC were harvested at month indicated after challenge with 1 × 10 8 FLK/BLV cells from indicated rabbits that had been infected with BLV SGV for 24 months or from age-matched naïve controls. DNA from 50,000 PMBC was subjected to nested PCR and gel electrophoresis and stained with ethidium bromide. The position of the 591-bp amplicon is designated by the labeled arrow. In parallel, DNA from 20,000 PBMC was subjected to PCR with primers complementary to β- actin . The position of the 594-bp amplicon is designated by the labeled arrow and the signals were quantified by densitometry.

    Article Snippet: To prepare peripheral blood mononuclear cell (PBMC) samples for PCR, PBMCs were isolated by a Ficoll-Paque (Pharmacia) gradient centrifugation and washed three times with phosphate buffered saline.

    Techniques: Real-time Polymerase Chain Reaction, Infection, Nested PCR, Nucleic Acid Electrophoresis, Staining, Amplification, Labeling, Polymerase Chain Reaction

    Semiquantitative PCR analysis to evaluate differences in proviral load. (A) PCR to detect BLV pol in PBMC from treated rabbits. Rabbit PBMC were harvested at 1, 4, and 10 months postinoculation and subjected to PCR with BLV pol primers KB560 and KB561 (10 × 10 4 PBMC) or β- actin to control for sample variation (2 × 10 4 PBMC). Each panel is labeled with month of sample harvest. Lanes are labeled with the source of PBMC DNA by rabbit number and treatment, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). In lower panels, the corresponding rabbit samples are designated by the matching parallel black lines. Positions of BLV pol amplicon (591 bp) and β- actin amplicon (594 bp) are designated. *, not determined (ND) because sample was not available; †, not determined because the animal died before harvest. (B) PCR standard curve. PBMC lysate harvested at 1 month postinoculation from BLV rabbit 44-10 was serially diluted in a range of 5 × 10 2 to 5 × 10 4 PBMC and subjected to PCR with BLV pol primers KB560 and KB561. Each lane is labeled with the number of cells used for PCR amplification, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). The arrow on the left indicates the position of BLV pol amplicon (591 bp), and the lines on the right indicate positions of 676- and 571-bp DNA size markers.

    Journal: Journal of Virology

    Article Title: Bovine Leukemia Virus Structural Gene Vectors Are Immunogenic and Lack Pathogenicity in a Rabbit Model

    doi:

    Figure Lengend Snippet: Semiquantitative PCR analysis to evaluate differences in proviral load. (A) PCR to detect BLV pol in PBMC from treated rabbits. Rabbit PBMC were harvested at 1, 4, and 10 months postinoculation and subjected to PCR with BLV pol primers KB560 and KB561 (10 × 10 4 PBMC) or β- actin to control for sample variation (2 × 10 4 PBMC). Each panel is labeled with month of sample harvest. Lanes are labeled with the source of PBMC DNA by rabbit number and treatment, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). In lower panels, the corresponding rabbit samples are designated by the matching parallel black lines. Positions of BLV pol amplicon (591 bp) and β- actin amplicon (594 bp) are designated. *, not determined (ND) because sample was not available; †, not determined because the animal died before harvest. (B) PCR standard curve. PBMC lysate harvested at 1 month postinoculation from BLV rabbit 44-10 was serially diluted in a range of 5 × 10 2 to 5 × 10 4 PBMC and subjected to PCR with BLV pol primers KB560 and KB561. Each lane is labeled with the number of cells used for PCR amplification, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). The arrow on the left indicates the position of BLV pol amplicon (591 bp), and the lines on the right indicate positions of 676- and 571-bp DNA size markers.

    Article Snippet: To prepare PBMCs samples for PCR, PBMCs were isolated by Ficoll-Paque (Pharmacia) gradient centrifugation and washed three times with phosphate-buffered saline.

    Techniques: Polymerase Chain Reaction, Labeling, Positive Control, Marker, Amplification

    The total number of circulating fibrocytes is unaffected by the leukocyte isolation strategy used. a Total number of circulating fibrocytes (defined as CD45 + lin − CD15 − CXCR4 + Col-1 + cells) per ml blood using two common leukocyte isolation techniques, e.g. the simple Pasteur pipette tube technique to isolate all white blood cells and Ficoll separation technique to isolate PBMCs. For this experiment we analyzed paired total white blood cells and PBMCs on the same day as blood withdrawal of 9 patients (4 IPF patients (black) and 5 PH patients (purple)) and 5 healthy controls (green). b Percentage of fibrocytes (of CD45 + cells) in the same patients after isolating all white blood cells (gray) and PBMCs (black). Data are depicted as median and interquartile

    Journal: Respiratory Research

    Article Title: Fibrocytes are increased in lung and peripheral blood of patients with idiopathic pulmonary fibrosis

    doi: 10.1186/s12931-018-0798-8

    Figure Lengend Snippet: The total number of circulating fibrocytes is unaffected by the leukocyte isolation strategy used. a Total number of circulating fibrocytes (defined as CD45 + lin − CD15 − CXCR4 + Col-1 + cells) per ml blood using two common leukocyte isolation techniques, e.g. the simple Pasteur pipette tube technique to isolate all white blood cells and Ficoll separation technique to isolate PBMCs. For this experiment we analyzed paired total white blood cells and PBMCs on the same day as blood withdrawal of 9 patients (4 IPF patients (black) and 5 PH patients (purple)) and 5 healthy controls (green). b Percentage of fibrocytes (of CD45 + cells) in the same patients after isolating all white blood cells (gray) and PBMCs (black). Data are depicted as median and interquartile

    Article Snippet: Briefly, following Ficoll density centrifugation, 2 × 10^5 PBMCs were plated into culture-slides (sigma, C7182, 0.8 cm2 /well) in complete culture medium (Dulbecco’s modified Eagle’s medium) (DMEM) supplemented with 20% fetal calf serum, 2 mM L-glutamine, 100 U/mL of penicillin, 100 mg/mL of streptomycin) (Life Technologies, Grand Island, NY) at 37 °C and 5% CO2.

    Techniques: Isolation, Transferring

    EAT-2 over-expression enhances human DC maturation. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-EAT2 or rAd5-Null. At 72 hpi, cells were stained for surface expression of CD86 (A), HLA-DR (B), CD80 (C) and CD83 (D) and FACS analysis was performed. Data are representative of at least three independent experiments from three different blood donors with similar results. Samples were plated in quadruplicates. Bars represent mean ± SD. Statistical analysis was completed using a one way ANOVA with a Student–Newman–Keuls post-hoc test. P

    Journal: International Immunology

    Article Title: Manipulation of EAT-2 expression promotes induction of multiple beneficial regulatory and effector functions of the human innate immune system as a novel immunomodulatory strategy

    doi: 10.1093/intimm/dxt061

    Figure Lengend Snippet: EAT-2 over-expression enhances human DC maturation. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-EAT2 or rAd5-Null. At 72 hpi, cells were stained for surface expression of CD86 (A), HLA-DR (B), CD80 (C) and CD83 (D) and FACS analysis was performed. Data are representative of at least three independent experiments from three different blood donors with similar results. Samples were plated in quadruplicates. Bars represent mean ± SD. Statistical analysis was completed using a one way ANOVA with a Student–Newman–Keuls post-hoc test. P

    Article Snippet: To identify human immune cells transduced by Ad-mediated EAT-2 or GFP gene transduction, human PBMCs (2×106 ) were infected with multiplicity of infection (MOI) of 10000 of Ad5-EAT-2 or Ad5-GFP for 48h and were then stained with the following antibodies: APC-Cy7-CD14, PE-CD1a, Alexa Fluor 700-CD8a, V450-CD4, APC-CD65 and PerCpCy5.5-CD3 (all at 4 µg ml−1 ; BD Biosciences).

    Techniques: Over Expression, Isolation, Infection, Staining, Expressing, FACS

    Transduction of human innate and adaptive immune cells by GFP-expressing rAd5 vector. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-GFP. FACS analysis for GFP expression at 48h following rAd5-GFP infection of human NK cells (A), CD4 + and CD8 + T cells (B), CD14 + monocytes and CD1a + cells (C) is shown. Data were collected using a BD LSRII flow cytometer and analyzed using FlowJo software.

    Journal: International Immunology

    Article Title: Manipulation of EAT-2 expression promotes induction of multiple beneficial regulatory and effector functions of the human innate immune system as a novel immunomodulatory strategy

    doi: 10.1093/intimm/dxt061

    Figure Lengend Snippet: Transduction of human innate and adaptive immune cells by GFP-expressing rAd5 vector. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-GFP. FACS analysis for GFP expression at 48h following rAd5-GFP infection of human NK cells (A), CD4 + and CD8 + T cells (B), CD14 + monocytes and CD1a + cells (C) is shown. Data were collected using a BD LSRII flow cytometer and analyzed using FlowJo software.

    Article Snippet: To identify human immune cells transduced by Ad-mediated EAT-2 or GFP gene transduction, human PBMCs (2×106 ) were infected with multiplicity of infection (MOI) of 10000 of Ad5-EAT-2 or Ad5-GFP for 48h and were then stained with the following antibodies: APC-Cy7-CD14, PE-CD1a, Alexa Fluor 700-CD8a, V450-CD4, APC-CD65 and PerCpCy5.5-CD3 (all at 4 µg ml−1 ; BD Biosciences).

    Techniques: Transduction, Expressing, Plasmid Preparation, Isolation, Infection, FACS, Flow Cytometry, Cytometry, Software

    Intracellular staining analysis for EAT-2 expression in human immune cells following rAd5-EAT2 infection. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-EAT2 or rAd5-EAT2(R31Q). Intracellular staining analysis for EAT-2 expression at 48h following Ad infection of human NK cells (A), CD14 + monocytes (B) and CD1a + DCs (C) is shown. Data were collected using a BD LSRII flow cytometer and analyzed using FlowJo software.

    Journal: International Immunology

    Article Title: Manipulation of EAT-2 expression promotes induction of multiple beneficial regulatory and effector functions of the human innate immune system as a novel immunomodulatory strategy

    doi: 10.1093/intimm/dxt061

    Figure Lengend Snippet: Intracellular staining analysis for EAT-2 expression in human immune cells following rAd5-EAT2 infection. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-EAT2 or rAd5-EAT2(R31Q). Intracellular staining analysis for EAT-2 expression at 48h following Ad infection of human NK cells (A), CD14 + monocytes (B) and CD1a + DCs (C) is shown. Data were collected using a BD LSRII flow cytometer and analyzed using FlowJo software.

    Article Snippet: To identify human immune cells transduced by Ad-mediated EAT-2 or GFP gene transduction, human PBMCs (2×106 ) were infected with multiplicity of infection (MOI) of 10000 of Ad5-EAT-2 or Ad5-GFP for 48h and were then stained with the following antibodies: APC-Cy7-CD14, PE-CD1a, Alexa Fluor 700-CD8a, V450-CD4, APC-CD65 and PerCpCy5.5-CD3 (all at 4 µg ml−1 ; BD Biosciences).

    Techniques: Staining, Expressing, Infection, Isolation, Flow Cytometry, Cytometry, Software

    EAT-2 over-expression enhances the cytolytic activity of human NK cells. Human PBMCs isolated from fresh buffy coat material were either mock infected or infected with MOI of 10000 with rAd5-EAT2, rAd5-EAT2(R31Q) or rAd5-Null for 72h and then co-cultured with CFSE-labeled K562 cells for an additional 48h at an E:T ratio of 1:10. Viability of K562 cells was assessed by either PI (A and B) or ViViD (C). Data are representative of two (A) or three (B) independent experiments from three different blood donors with similar results. Samples were plated in quadruplicates. Bars represent mean ± SD. Statistical analysis was completed using a one way ANOVA with a Student–Newman–Keuls post-hoc test. P

    Journal: International Immunology

    Article Title: Manipulation of EAT-2 expression promotes induction of multiple beneficial regulatory and effector functions of the human innate immune system as a novel immunomodulatory strategy

    doi: 10.1093/intimm/dxt061

    Figure Lengend Snippet: EAT-2 over-expression enhances the cytolytic activity of human NK cells. Human PBMCs isolated from fresh buffy coat material were either mock infected or infected with MOI of 10000 with rAd5-EAT2, rAd5-EAT2(R31Q) or rAd5-Null for 72h and then co-cultured with CFSE-labeled K562 cells for an additional 48h at an E:T ratio of 1:10. Viability of K562 cells was assessed by either PI (A and B) or ViViD (C). Data are representative of two (A) or three (B) independent experiments from three different blood donors with similar results. Samples were plated in quadruplicates. Bars represent mean ± SD. Statistical analysis was completed using a one way ANOVA with a Student–Newman–Keuls post-hoc test. P

    Article Snippet: To identify human immune cells transduced by Ad-mediated EAT-2 or GFP gene transduction, human PBMCs (2×106 ) were infected with multiplicity of infection (MOI) of 10000 of Ad5-EAT-2 or Ad5-GFP for 48h and were then stained with the following antibodies: APC-Cy7-CD14, PE-CD1a, Alexa Fluor 700-CD8a, V450-CD4, APC-CD65 and PerCpCy5.5-CD3 (all at 4 µg ml−1 ; BD Biosciences).

    Techniques: Over Expression, Activity Assay, Isolation, Infection, Cell Culture, Labeling

    Frequency and molecular expression of pDC in IT, CHB and AHB groups. (a) AHB group had significantly higher PBMC percent in peripheral blood leukocytes, compared with CHB or IT group. (b) Frequency of pDC in PBMC decreased in AHB and CHB groups, compared with IT group. (c) The difference in CD86+ pDC frequency among three groups was not significant. (d) The counts of CD86 molecular expressed on surface of pDC in CHB group was higher, compared with AHB and IT groups. IT group: HBeAg-positive chronic hepatitis B virus infected patients in immune tolerance phase; CHB group: HBeAg-positive chronic hepatitis B infected patients; AHB group: Acute hepatitis B virus infected patients; PBMC: Peripheral blood mononuclear cells; pDC: Plasmacytoid dendritic cell; CD86: Cluster of differentiation antigen 86.

    Journal: Chinese Medical Journal

    Article Title: Plasmacytoid Dendritic Cell Function and Cytokine Network Profiles in Patients with Acute or Chronic Hepatitis B Virus Infection

    doi: 10.4103/0366-6999.221275

    Figure Lengend Snippet: Frequency and molecular expression of pDC in IT, CHB and AHB groups. (a) AHB group had significantly higher PBMC percent in peripheral blood leukocytes, compared with CHB or IT group. (b) Frequency of pDC in PBMC decreased in AHB and CHB groups, compared with IT group. (c) The difference in CD86+ pDC frequency among three groups was not significant. (d) The counts of CD86 molecular expressed on surface of pDC in CHB group was higher, compared with AHB and IT groups. IT group: HBeAg-positive chronic hepatitis B virus infected patients in immune tolerance phase; CHB group: HBeAg-positive chronic hepatitis B infected patients; AHB group: Acute hepatitis B virus infected patients; PBMC: Peripheral blood mononuclear cells; pDC: Plasmacytoid dendritic cell; CD86: Cluster of differentiation antigen 86.

    Article Snippet: Frequency and molecular expression of plasmacytoid dendritic cell In this study, the peripheral blood mononuclear cells (PBMCs) percentage of peripheral blood leukocytes, frequency of pDC in PBMC, frequency of cluster of differentiation antigen 86 (CD86) + pDC, and the counts of CD86 molecular expressed on surface of pDC were measured by four-color flow cytometry (FACS Caliburflow Cytometer; Becton-Dickinson, USA); and the steps to measure the expression of pDCs were as follows: the 100 μl of whole peripheral blood samples were incubated with monoclonal antibodies (mAbs) of Lin1-fluorescein isothiocyanate, human leukocyte antigen DR (HLA-DR)-peridinin chlorophyll protein, CD123-human antigen presenting cells, and CD86 - phycoerythrin (PE; all provided by BD Biosciences, Cowley, UK) inappropriate tubes at room temperature in the dark for 20 min, then added 2 ml of fluorescence activating cell sorter (FACS) Lysing solution and incubated at room temperature in the dark for 5 min, centrifuged at 300 ×g for 5 min, aspirated the supernatant, then added 2 ml of phosphate buffer saline (PBS) and vortex gently, centrifuged at 300 ×g for 5 min and aspirated the supernatant again, vortex gently and re-suspended with 200 μl PBS; finally analyzed using the FACS flow cytometer. pDCs were identified as mononuclear cells that the Lin1-(CD3-CD14-CD16-CD19-CD20-)/HLA-DR+/CD123+, the activation/maturation state of pDC was assessed by the co-stimulatory molecules CD86 in Lin1-CD123+ HLA-DR+cells.

    Techniques: Expressing, Infection

    Frequency and molecular expression of pDC in HI and HBV infected groups. (a) The difference in percentages of PBMC in peripheral blood leukocytes between two groups was not significant. Compared with HI group, frequency of pDC in PBMC was significantly lower (b), but CD86+ pDC frequency and counts of CD86 molecular expressed on surface of pDC were higher in HBV infected group (c and d). HI: Healthy individual; HBV: Hepatitis B virus; PBMC: Peripheral blood mononuclear cells; pDC: Plasmacytoid dendritic cell; CD86: Cluster of differentiation antigen 86.

    Journal: Chinese Medical Journal

    Article Title: Plasmacytoid Dendritic Cell Function and Cytokine Network Profiles in Patients with Acute or Chronic Hepatitis B Virus Infection

    doi: 10.4103/0366-6999.221275

    Figure Lengend Snippet: Frequency and molecular expression of pDC in HI and HBV infected groups. (a) The difference in percentages of PBMC in peripheral blood leukocytes between two groups was not significant. Compared with HI group, frequency of pDC in PBMC was significantly lower (b), but CD86+ pDC frequency and counts of CD86 molecular expressed on surface of pDC were higher in HBV infected group (c and d). HI: Healthy individual; HBV: Hepatitis B virus; PBMC: Peripheral blood mononuclear cells; pDC: Plasmacytoid dendritic cell; CD86: Cluster of differentiation antigen 86.

    Article Snippet: Frequency and molecular expression of plasmacytoid dendritic cell In this study, the peripheral blood mononuclear cells (PBMCs) percentage of peripheral blood leukocytes, frequency of pDC in PBMC, frequency of cluster of differentiation antigen 86 (CD86) + pDC, and the counts of CD86 molecular expressed on surface of pDC were measured by four-color flow cytometry (FACS Caliburflow Cytometer; Becton-Dickinson, USA); and the steps to measure the expression of pDCs were as follows: the 100 μl of whole peripheral blood samples were incubated with monoclonal antibodies (mAbs) of Lin1-fluorescein isothiocyanate, human leukocyte antigen DR (HLA-DR)-peridinin chlorophyll protein, CD123-human antigen presenting cells, and CD86 - phycoerythrin (PE; all provided by BD Biosciences, Cowley, UK) inappropriate tubes at room temperature in the dark for 20 min, then added 2 ml of fluorescence activating cell sorter (FACS) Lysing solution and incubated at room temperature in the dark for 5 min, centrifuged at 300 ×g for 5 min, aspirated the supernatant, then added 2 ml of phosphate buffer saline (PBS) and vortex gently, centrifuged at 300 ×g for 5 min and aspirated the supernatant again, vortex gently and re-suspended with 200 μl PBS; finally analyzed using the FACS flow cytometer. pDCs were identified as mononuclear cells that the Lin1-(CD3-CD14-CD16-CD19-CD20-)/HLA-DR+/CD123+, the activation/maturation state of pDC was assessed by the co-stimulatory molecules CD86 in Lin1-CD123+ HLA-DR+cells.

    Techniques: Expressing, Infection

    Exosomes from tumor patients with increased membrane-associated HSP72 and HSP105 induced DCs to promote tumor cell invasion via IL-6 in vitro . (A) After adsorption onto latex beads, HSP72 and HSP105 on EXO-PT and EXO-TT were detected by flow cytometry. RFI was calculated by dividing mean fluorescence intensity of samples staining with detection Abs by that of sample staining with ISO Abs (n = 7). (B) Human DCs were induced from PBMCs and stimulated with 5 μg/ml of EXO-PT or EXO-TT from breast tumor tissues for 6 h. The IL-6 production was measured by ELISA (n = 3). (C) MDA-MB-435S cells were cultured in supernatant from PBS-, EXO-PT- or EXO-TT (5 μg/ml for 6 h)-stimulated DCs with or without 30 μg/ml of anti-IL-6 or ISO mAbs for 24 h. Then, their invasive ability was measured using an in vitro invasive assay, and the cells on the bottom of the Transwell filter were imaged and quantified (Magnification: 200 ×). (D) The results of (C) were statistically analyzed (n = 5). (E, F) After adsorption onto latex beads, HSP72 and HSP105 on exosomes from sera of breast tumor patients or healthy controls were detected by flow cytometry. One representative result of 12 is shown (E). RFI was calculated and statistically analyzed (F). (B, D) The results are shown as the mean ± SEM of 3 independent experiments. (C) One representative of 3 independent experiments is shown. P values were generated by 2-tail student's t -test in A, F, and by one-way ANOVA, followed by a Tukey-Kramer multiple comparison test in B, D; *** p

    Journal: Oncoimmunology

    Article Title: Tumor-derived exosomes educate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway

    doi: 10.1080/2162402X.2017.1362527

    Figure Lengend Snippet: Exosomes from tumor patients with increased membrane-associated HSP72 and HSP105 induced DCs to promote tumor cell invasion via IL-6 in vitro . (A) After adsorption onto latex beads, HSP72 and HSP105 on EXO-PT and EXO-TT were detected by flow cytometry. RFI was calculated by dividing mean fluorescence intensity of samples staining with detection Abs by that of sample staining with ISO Abs (n = 7). (B) Human DCs were induced from PBMCs and stimulated with 5 μg/ml of EXO-PT or EXO-TT from breast tumor tissues for 6 h. The IL-6 production was measured by ELISA (n = 3). (C) MDA-MB-435S cells were cultured in supernatant from PBS-, EXO-PT- or EXO-TT (5 μg/ml for 6 h)-stimulated DCs with or without 30 μg/ml of anti-IL-6 or ISO mAbs for 24 h. Then, their invasive ability was measured using an in vitro invasive assay, and the cells on the bottom of the Transwell filter were imaged and quantified (Magnification: 200 ×). (D) The results of (C) were statistically analyzed (n = 5). (E, F) After adsorption onto latex beads, HSP72 and HSP105 on exosomes from sera of breast tumor patients or healthy controls were detected by flow cytometry. One representative result of 12 is shown (E). RFI was calculated and statistically analyzed (F). (B, D) The results are shown as the mean ± SEM of 3 independent experiments. (C) One representative of 3 independent experiments is shown. P values were generated by 2-tail student's t -test in A, F, and by one-way ANOVA, followed by a Tukey-Kramer multiple comparison test in B, D; *** p

    Article Snippet: To generate human DCs, peripheral blood mononuclear cells (PBMCs) from healthy volunteers were isolated by density centrifugation of heparinized blood on LSM® (MP Biomedicals), resuspended in culture medium and allowed to adhere in 6-well plates.

    Techniques: In Vitro, Adsorption, Flow Cytometry, Cytometry, Fluorescence, Staining, Enzyme-linked Immunosorbent Assay, Multiple Displacement Amplification, Cell Culture, Generated

    DCs with enhanced IL-6-secretion capacity were found in tumor-bearing mice and patients. (A, B) Mice were subcutaneously inoculated with 5 × 10 5 B16-F10 cells, and mice without tumor inoculation were used as controls. Then, DCs from splenocytes were isolated 1 week, 2 weeks or 3 weeks later and stimulated with PBS, 5 μg/ml B16-F10-EXO or 100 ng/ml of LPS for 6 h. IL-6 production by DCs was detected by ELISA (n = 3) (A). DCs from splenocytes were isolated 3 weeks later and cultured in vitro for 6 h. Culture supernatant was collected and B16-F10 cells were cultured in this supernatant for 24 h. Then, the invasive ability of B16-F10 cells was measured (n = 5). Control indicates B16-F10 cells without any treatment (B). SN-DC PBS or SN-DC tumor denotes supernatant from DCs of healthy control mice or tumor mice, respectively. (C) Mice were intravenously injected with 100 μl of PBS or 10 μg/100 μl of B16-F10-EXO. Then, DCs from splenocytes were isolated 12 or 24 h later and cultured in vitro for 6 h. IL-6 production by DCs was detected by ELISA (n = 3). (D) Human DCs were isolated from PBMCs from healthy volunteers (n = 27) or breast tumor patients (n = 18) and cultured in vitro for 6 h with or without 100 ng/ml LPS. IL-6 production by DCs was detected by ELISA. The results are shown as the mean ± SEM of 3 independent experiments or of all detected samples. P values were generated by a 2-tail student's t -test.

    Journal: Oncoimmunology

    Article Title: Tumor-derived exosomes educate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway

    doi: 10.1080/2162402X.2017.1362527

    Figure Lengend Snippet: DCs with enhanced IL-6-secretion capacity were found in tumor-bearing mice and patients. (A, B) Mice were subcutaneously inoculated with 5 × 10 5 B16-F10 cells, and mice without tumor inoculation were used as controls. Then, DCs from splenocytes were isolated 1 week, 2 weeks or 3 weeks later and stimulated with PBS, 5 μg/ml B16-F10-EXO or 100 ng/ml of LPS for 6 h. IL-6 production by DCs was detected by ELISA (n = 3) (A). DCs from splenocytes were isolated 3 weeks later and cultured in vitro for 6 h. Culture supernatant was collected and B16-F10 cells were cultured in this supernatant for 24 h. Then, the invasive ability of B16-F10 cells was measured (n = 5). Control indicates B16-F10 cells without any treatment (B). SN-DC PBS or SN-DC tumor denotes supernatant from DCs of healthy control mice or tumor mice, respectively. (C) Mice were intravenously injected with 100 μl of PBS or 10 μg/100 μl of B16-F10-EXO. Then, DCs from splenocytes were isolated 12 or 24 h later and cultured in vitro for 6 h. IL-6 production by DCs was detected by ELISA (n = 3). (D) Human DCs were isolated from PBMCs from healthy volunteers (n = 27) or breast tumor patients (n = 18) and cultured in vitro for 6 h with or without 100 ng/ml LPS. IL-6 production by DCs was detected by ELISA. The results are shown as the mean ± SEM of 3 independent experiments or of all detected samples. P values were generated by a 2-tail student's t -test.

    Article Snippet: To generate human DCs, peripheral blood mononuclear cells (PBMCs) from healthy volunteers were isolated by density centrifugation of heparinized blood on LSM® (MP Biomedicals), resuspended in culture medium and allowed to adhere in 6-well plates.

    Techniques: Mouse Assay, Isolation, Enzyme-linked Immunosorbent Assay, Cell Culture, In Vitro, Injection, Generated

    Unchanged healthy donor NK cell degranulation in response to GAD65 AA 114–122 peptide-pulsed APCs. Degranulation of CD3 - CD56 + ILT2 + NK cells of PBMC from HD patients, expanded with GAD65 AA 114–122 peptide, measured as CD107a cell-surface expression following stimulation with APCs, either left unpulsed or GAD65 AA 114–122 peptide-pulsed. A representative experiment out of two performed is shown. K562 cells were used as positive control. The percentage of CD3 - CD56 + CD107a + NK cells (upper panel) and of CD3 - CD56 + CD107a + ILT2 + (lower panel) is indicated for each condition.

    Journal: PLoS ONE

    Article Title: Identification of GAD65 AA 114-122 reactive 'memory-like' NK cells in newly diagnosed Type 1 diabetic patients by HLA-class I pentamers

    doi: 10.1371/journal.pone.0189615

    Figure Lengend Snippet: Unchanged healthy donor NK cell degranulation in response to GAD65 AA 114–122 peptide-pulsed APCs. Degranulation of CD3 - CD56 + ILT2 + NK cells of PBMC from HD patients, expanded with GAD65 AA 114–122 peptide, measured as CD107a cell-surface expression following stimulation with APCs, either left unpulsed or GAD65 AA 114–122 peptide-pulsed. A representative experiment out of two performed is shown. K562 cells were used as positive control. The percentage of CD3 - CD56 + CD107a + NK cells (upper panel) and of CD3 - CD56 + CD107a + ILT2 + (lower panel) is indicated for each condition.

    Article Snippet: Co-culture On day 6 PBMC, either GAD65 AA 114–122 or FLU peptides stimulated for 4 days, additionally cultured for two days in the presence of IL-2 (100 IU/ml), were co-cultured with pulsed or unpulsed APCs at 1:3 (PBMC:APCs) ratio in 96 well round bottomed culture plates (Corning Incorporated, Corning, NY 14831–001, USA) for 2 and half hours in RPMI 10% FBS complete medium (see above ) additionally supplemented with GolgiStop reagent (1:500 dilution, BD Biosciences).

    Techniques: Expressing, Positive Control

    Increased susceptibility of GAD65 AA 114–122 peptide-pulsed APCs to T1D NK cell-mediated recognition associates with NK cell-ILT2 expression. Degranulation of CD3 - CD56 + ILT2 + NK cells of PBMC from T1D patients, expanded with GAD65 AA 114–122 or FLU peptides, measured as CD107a cell-surface expression following stimulation with APCs, either left unpulsed or GAD65 AA 114–122 peptide-pulsed. (A) A representative experiment out of two performed is shown. K562 cells were used as positive control. The percentage of CD3 - CD56 + CD107a + NK cells (upper panel) and CD3 - CD56 + CD107a + ILT2 + (lower panel) is indicated for each condition. (B) Summary of CD3 - CD56 + CD107a + and (C) CD3 - CD56 + ILT2 + CD107a + NK cell percentage of four T1D PBMC, expanded with GAD65 AA 114–122 or FLU peptides, following stimulation with APCs, either left unpulsed (circle dots) or GAD65 AA 114–122 (GAD65)-pulsed (square dots); horizontal bars, average values are reported.

    Journal: PLoS ONE

    Article Title: Identification of GAD65 AA 114-122 reactive 'memory-like' NK cells in newly diagnosed Type 1 diabetic patients by HLA-class I pentamers

    doi: 10.1371/journal.pone.0189615

    Figure Lengend Snippet: Increased susceptibility of GAD65 AA 114–122 peptide-pulsed APCs to T1D NK cell-mediated recognition associates with NK cell-ILT2 expression. Degranulation of CD3 - CD56 + ILT2 + NK cells of PBMC from T1D patients, expanded with GAD65 AA 114–122 or FLU peptides, measured as CD107a cell-surface expression following stimulation with APCs, either left unpulsed or GAD65 AA 114–122 peptide-pulsed. (A) A representative experiment out of two performed is shown. K562 cells were used as positive control. The percentage of CD3 - CD56 + CD107a + NK cells (upper panel) and CD3 - CD56 + CD107a + ILT2 + (lower panel) is indicated for each condition. (B) Summary of CD3 - CD56 + CD107a + and (C) CD3 - CD56 + ILT2 + CD107a + NK cell percentage of four T1D PBMC, expanded with GAD65 AA 114–122 or FLU peptides, following stimulation with APCs, either left unpulsed (circle dots) or GAD65 AA 114–122 (GAD65)-pulsed (square dots); horizontal bars, average values are reported.

    Article Snippet: Co-culture On day 6 PBMC, either GAD65 AA 114–122 or FLU peptides stimulated for 4 days, additionally cultured for two days in the presence of IL-2 (100 IU/ml), were co-cultured with pulsed or unpulsed APCs at 1:3 (PBMC:APCs) ratio in 96 well round bottomed culture plates (Corning Incorporated, Corning, NY 14831–001, USA) for 2 and half hours in RPMI 10% FBS complete medium (see above ) additionally supplemented with GolgiStop reagent (1:500 dilution, BD Biosciences).

    Techniques: Expressing, Positive Control

    Pre-transplant CMV-specific T cell responses in the patients with CMV episode. CMV-specific T cell responses were measured by stimulating PBMCs with OLPs pools of (A) pp65, (B) IE-1 in ELISPOT or (D) combination of immunodominant peptides of CMV in QuantiFERON-CMV. The combined result of (A) and (B) is represented in (C) pp65 or IE-1. In (A-C), pre-transplant CMV-specific T cell responses of the patients with post-transplant CMV episode were likely to be higher than the patients without post-transplant CMV episode. In (D), however, IFN-γ concentration was tended to lower in the patients with CMV infection. Each result was obtained by 2 repeated experiments. CMV, cytomegalovirus; PBMC, peripheral blood mononuclear cell; OLP, overlapping peptide; pp65, phosphoprotein 65; IE-1, immediate-early 1; ELISPOT, enzyme-linked immunospot; IFN, interferon.

    Journal: Immune Network

    Article Title: Comparison of the Commercial QuantiFERON-CMV and Overlapping Peptide-based ELISPOT Assays for Predicting CMV Infection in Kidney Transplant Recipients

    doi: 10.4110/in.2017.17.5.317

    Figure Lengend Snippet: Pre-transplant CMV-specific T cell responses in the patients with CMV episode. CMV-specific T cell responses were measured by stimulating PBMCs with OLPs pools of (A) pp65, (B) IE-1 in ELISPOT or (D) combination of immunodominant peptides of CMV in QuantiFERON-CMV. The combined result of (A) and (B) is represented in (C) pp65 or IE-1. In (A-C), pre-transplant CMV-specific T cell responses of the patients with post-transplant CMV episode were likely to be higher than the patients without post-transplant CMV episode. In (D), however, IFN-γ concentration was tended to lower in the patients with CMV infection. Each result was obtained by 2 repeated experiments. CMV, cytomegalovirus; PBMC, peripheral blood mononuclear cell; OLP, overlapping peptide; pp65, phosphoprotein 65; IE-1, immediate-early 1; ELISPOT, enzyme-linked immunospot; IFN, interferon.

    Article Snippet: OLPs-based ELISPOT Peripheral blood was collected from each patient before transplantation, and peripheral blood mononuclear cells (PBMCs) were isolated using Lymphocyte Separation Medium (Corning, New York, NY, USA).

    Techniques: Enzyme-linked Immunospot, Concentration Assay, Infection

    Inference and effect of barcode multiplets in single-cell ATAC-seq data. (a) Default t-SNE depiction of public scATAC-seq PBMC 5k dataset. Colors represent cluster annotations from the automated CellRanger output. (b) Quantification of barcodes affected by barcode multiplets for the same dataset (identified by bap). (c) Depiction of two multiplets each composed of 9 oligonucleotide barcodes. Barcodes in each multiplet share a long common subsequence, denoted in black. (d) Visualization of two barcode multiplets from (c) in t-SNE coordinates. (e) Visualization of all implicated barcode multiplets from this dataset. The zoomed panel shows a small group of cells affected by five multiplets, indicated by color. (f) Empirical distribution of the mean restricted longest common subsequence (rLCS) per multiplet. A cutoff of 6 was used to determine either of the two classes of barcode multiplets. (g) Percent difference of the mean log2 fragments between pairs of barcodes within a multiplet. The reported p-value is from a two-sided Kolmogorov–Smirnov test. Boxplots: center line, median; box limits, first and third quartiles; whiskers, 1.5x interquartile range. (h) Overall rates of barcode multiplets from additional scATAC-seq data comparing v1.0 and v1.1 (NextGEM) chip designs.

    Journal: bioRxiv

    Article Title: Inference and effects of barcode multiplets in droplet-based single-cell assays

    doi: 10.1101/824003

    Figure Lengend Snippet: Inference and effect of barcode multiplets in single-cell ATAC-seq data. (a) Default t-SNE depiction of public scATAC-seq PBMC 5k dataset. Colors represent cluster annotations from the automated CellRanger output. (b) Quantification of barcodes affected by barcode multiplets for the same dataset (identified by bap). (c) Depiction of two multiplets each composed of 9 oligonucleotide barcodes. Barcodes in each multiplet share a long common subsequence, denoted in black. (d) Visualization of two barcode multiplets from (c) in t-SNE coordinates. (e) Visualization of all implicated barcode multiplets from this dataset. The zoomed panel shows a small group of cells affected by five multiplets, indicated by color. (f) Empirical distribution of the mean restricted longest common subsequence (rLCS) per multiplet. A cutoff of 6 was used to determine either of the two classes of barcode multiplets. (g) Percent difference of the mean log2 fragments between pairs of barcodes within a multiplet. The reported p-value is from a two-sided Kolmogorov–Smirnov test. Boxplots: center line, median; box limits, first and third quartiles; whiskers, 1.5x interquartile range. (h) Overall rates of barcode multiplets from additional scATAC-seq data comparing v1.0 and v1.1 (NextGEM) chip designs.

    Article Snippet: Profiling PBMCs using 10x scATAC-seq For 10x scATAC-seq experiments with PBMCs (PB003F, Allcells), frozen cells were quickly thawed in a 37°C water bath for about 30s and transferred to a 15 mL tube.

    Techniques: Chromatin Immunoprecipitation

    Supporting information for Figure 3 . (a) Quantification of barcodes affected by barcode multiplets for the PBMC dataset generated with this work (“This Study”). (b) Percentage of barcode multiplets identified for different numbers of input barcodes (see Methods ). (c) Visualization of seven additional barcode multiplets from the Public dataset. (d) Proportion of bead pairs occurring in the same chromatin accessibility-defined Louvain cluster compared to a permuted background. Error bars represent standard error of mean over 100 permutations per dataset. (e) Downsampling analysis of the dataset generated in this work (“This Study”). Barcode multiplets were examined at downsampled intervals from 10%-90% by units of 10%. The highlighted sample represents 40% downsampling and corresponds to a median 10,000 fragments detected per barcode. At all downsampled thresholds, we detected 0 pairs that were not present in the 100% sample. (f) Distribution of the restricted longest common subsequence (rLCS) for 1,000,000 randomly-sampled barcode pairs in the 10x barcode universe. A threshold at 6 is drawn for use in other analyses.

    Journal: bioRxiv

    Article Title: Inference and effects of barcode multiplets in droplet-based single-cell assays

    doi: 10.1101/824003

    Figure Lengend Snippet: Supporting information for Figure 3 . (a) Quantification of barcodes affected by barcode multiplets for the PBMC dataset generated with this work (“This Study”). (b) Percentage of barcode multiplets identified for different numbers of input barcodes (see Methods ). (c) Visualization of seven additional barcode multiplets from the Public dataset. (d) Proportion of bead pairs occurring in the same chromatin accessibility-defined Louvain cluster compared to a permuted background. Error bars represent standard error of mean over 100 permutations per dataset. (e) Downsampling analysis of the dataset generated in this work (“This Study”). Barcode multiplets were examined at downsampled intervals from 10%-90% by units of 10%. The highlighted sample represents 40% downsampling and corresponds to a median 10,000 fragments detected per barcode. At all downsampled thresholds, we detected 0 pairs that were not present in the 100% sample. (f) Distribution of the restricted longest common subsequence (rLCS) for 1,000,000 randomly-sampled barcode pairs in the 10x barcode universe. A threshold at 6 is drawn for use in other analyses.

    Article Snippet: Profiling PBMCs using 10x scATAC-seq For 10x scATAC-seq experiments with PBMCs (PB003F, Allcells), frozen cells were quickly thawed in a 37°C water bath for about 30s and transferred to a 15 mL tube.

    Techniques: Generated

    ( a ) p24 viral antigen levels 10 days post-infection in the supernatant of HIV-1 infected PBMCs treated with TIZ (10 μg/mL) or RM-4848 (10 μg/mL). Results obtained with PBMC of 20 different healthy donors are shown. Median values and statistical significance is presented. ( b ) Inhibition (%) of viral replication in PBMC of 20 different healthy donors infected in vitro with HIV-1 in the presence of TIZ- (10 μg/mL) or RM-4848- (10 μg/mL). Median values and statistical significance is presented. ( c ) p24 viral antigen levels in PBMCs infected with HIV-1 in the presence of RM-4848 throughout infection (RM-4848)(10 μg/mL) or in medium alone (no treatment). In addition to these two situations, results of 3 other experimental conditions are shown: 1) PBMCs treated with RM-4848 before being infected (pre-infection); 2) RM-4848 present during the 24 hours infection period (during infection) and then washed away; 3) RM-4848 added 24 hours after infection (post infection). Mean values ± standard errors are shown.

    Journal: Scientific Reports

    Article Title: Thiazolides Elicit Anti-Viral Innate Immunity and Reduce HIV Replication

    doi: 10.1038/srep27148

    Figure Lengend Snippet: ( a ) p24 viral antigen levels 10 days post-infection in the supernatant of HIV-1 infected PBMCs treated with TIZ (10 μg/mL) or RM-4848 (10 μg/mL). Results obtained with PBMC of 20 different healthy donors are shown. Median values and statistical significance is presented. ( b ) Inhibition (%) of viral replication in PBMC of 20 different healthy donors infected in vitro with HIV-1 in the presence of TIZ- (10 μg/mL) or RM-4848- (10 μg/mL). Median values and statistical significance is presented. ( c ) p24 viral antigen levels in PBMCs infected with HIV-1 in the presence of RM-4848 throughout infection (RM-4848)(10 μg/mL) or in medium alone (no treatment). In addition to these two situations, results of 3 other experimental conditions are shown: 1) PBMCs treated with RM-4848 before being infected (pre-infection); 2) RM-4848 present during the 24 hours infection period (during infection) and then washed away; 3) RM-4848 added 24 hours after infection (post infection). Mean values ± standard errors are shown.

    Article Snippet: PBMC isolation and HIV-1 infection Whole blood was collected from 20 healthy volunteers by venipuncture in Vacutainer tubes containing EDTA (Becton Dickinson, Franklin Lakes, NJ), and PBMCs were separated on lymphocyte separation medium (Organon Teknica, Malvern, PA).

    Techniques: Infection, Inhibition, In Vitro

    Cytokines (IL-2, IFNγ) and chemokines (MCP-1, MIP-1α, MIP-1β, RANTES) production (pg/ml) by PBMCs of healthy donors. PBMCs were either uninfected (white bars) or HIV-1-infected (grey bars) and were cultured in medium alone (no drug), or in medium containing either TIZ- (10 μg/mL) ( a ) or RM-4848- (10 μg/mL) ( b ). Mean values 7 days post-infection ± standard errors and statistical differences are shown.

    Journal: Scientific Reports

    Article Title: Thiazolides Elicit Anti-Viral Innate Immunity and Reduce HIV Replication

    doi: 10.1038/srep27148

    Figure Lengend Snippet: Cytokines (IL-2, IFNγ) and chemokines (MCP-1, MIP-1α, MIP-1β, RANTES) production (pg/ml) by PBMCs of healthy donors. PBMCs were either uninfected (white bars) or HIV-1-infected (grey bars) and were cultured in medium alone (no drug), or in medium containing either TIZ- (10 μg/mL) ( a ) or RM-4848- (10 μg/mL) ( b ). Mean values 7 days post-infection ± standard errors and statistical differences are shown.

    Article Snippet: PBMC isolation and HIV-1 infection Whole blood was collected from 20 healthy volunteers by venipuncture in Vacutainer tubes containing EDTA (Becton Dickinson, Franklin Lakes, NJ), and PBMCs were separated on lymphocyte separation medium (Organon Teknica, Malvern, PA).

    Techniques: Infection, Cell Culture

    mRNA expression of 84 genes that are part of the HIV-1 life cycle and known to directly obstacle HIV infectivity has been assessed by real-time quantitative RT-PCR, calculated relative to five housekeeping genes and shown as fold-change expression from the untreated sample. Gene expression (nfold) is shown as a color scale from green to red (−2 to +9) (MEV multiple experiment viewer software). Results were obtained in HIV1-infected PBMCs at day 7 post infection, cultured in medium containing TIZ (10 μg/mL) or RM-4848 (10 μg/mL). Only targets showing > 2-fold modulation are considered significant and are shown in tab.

    Journal: Scientific Reports

    Article Title: Thiazolides Elicit Anti-Viral Innate Immunity and Reduce HIV Replication

    doi: 10.1038/srep27148

    Figure Lengend Snippet: mRNA expression of 84 genes that are part of the HIV-1 life cycle and known to directly obstacle HIV infectivity has been assessed by real-time quantitative RT-PCR, calculated relative to five housekeeping genes and shown as fold-change expression from the untreated sample. Gene expression (nfold) is shown as a color scale from green to red (−2 to +9) (MEV multiple experiment viewer software). Results were obtained in HIV1-infected PBMCs at day 7 post infection, cultured in medium containing TIZ (10 μg/mL) or RM-4848 (10 μg/mL). Only targets showing > 2-fold modulation are considered significant and are shown in tab.

    Article Snippet: PBMC isolation and HIV-1 infection Whole blood was collected from 20 healthy volunteers by venipuncture in Vacutainer tubes containing EDTA (Becton Dickinson, Franklin Lakes, NJ), and PBMCs were separated on lymphocyte separation medium (Organon Teknica, Malvern, PA).

    Techniques: Expressing, Infection, Quantitative RT-PCR, Software, Cell Culture

    mRNA expression of genes involved in cholesterol metabolism and efflux in PBMCs of healthy donors. ( a ) Uninfected PBMCs cultured in medium containing TIZ (10 μg/mL); ( b ) Uninfected PBMCs cultured in medium containing RM-4848 (10 μg/mL); ( c ) HIV-1–infected PBMCs (day 7 post infection) cultured in medium containing TIZ (10 μg/mL); ( d ) HIV-1–infected PBMCs (day 7 post infection) cultured in medium containing RM-4848 (10 μg/mL). Gene expression genes is assessed by quantitative RT-PCR and shown as fold-change expression from the untreated sample. Only targets showing > 2-fold modulation are considered significant. Mean values ± S.D. and p values are indicated.

    Journal: Scientific Reports

    Article Title: Thiazolides Elicit Anti-Viral Innate Immunity and Reduce HIV Replication

    doi: 10.1038/srep27148

    Figure Lengend Snippet: mRNA expression of genes involved in cholesterol metabolism and efflux in PBMCs of healthy donors. ( a ) Uninfected PBMCs cultured in medium containing TIZ (10 μg/mL); ( b ) Uninfected PBMCs cultured in medium containing RM-4848 (10 μg/mL); ( c ) HIV-1–infected PBMCs (day 7 post infection) cultured in medium containing TIZ (10 μg/mL); ( d ) HIV-1–infected PBMCs (day 7 post infection) cultured in medium containing RM-4848 (10 μg/mL). Gene expression genes is assessed by quantitative RT-PCR and shown as fold-change expression from the untreated sample. Only targets showing > 2-fold modulation are considered significant. Mean values ± S.D. and p values are indicated.

    Article Snippet: PBMC isolation and HIV-1 infection Whole blood was collected from 20 healthy volunteers by venipuncture in Vacutainer tubes containing EDTA (Becton Dickinson, Franklin Lakes, NJ), and PBMCs were separated on lymphocyte separation medium (Organon Teknica, Malvern, PA).

    Techniques: Expressing, Cell Culture, Infection, Quantitative RT-PCR

    p24 viral antigen concentration in HIV-1 - infected PBMCs cultured in medium alone (no drug) or in the presence of TO-901317 (1 μM), an LXR agonist. Results were obtained with PBMCs of 20 different healthy donors 10 days post-infection. Mean values ± standard errors and statistical significance are shown.

    Journal: Scientific Reports

    Article Title: Thiazolides Elicit Anti-Viral Innate Immunity and Reduce HIV Replication

    doi: 10.1038/srep27148

    Figure Lengend Snippet: p24 viral antigen concentration in HIV-1 - infected PBMCs cultured in medium alone (no drug) or in the presence of TO-901317 (1 μM), an LXR agonist. Results were obtained with PBMCs of 20 different healthy donors 10 days post-infection. Mean values ± standard errors and statistical significance are shown.

    Article Snippet: PBMC isolation and HIV-1 infection Whole blood was collected from 20 healthy volunteers by venipuncture in Vacutainer tubes containing EDTA (Becton Dickinson, Franklin Lakes, NJ), and PBMCs were separated on lymphocyte separation medium (Organon Teknica, Malvern, PA).

    Techniques: Concentration Assay, Infection, Cell Culture

    Rituximab ( RTX ) leads to further phenotypical changes in CD56 dim natural killer ( NK ) cells. Freshly isolated peripheral blood mononuclear cells (PBMCs) were incubated with (+) or without ( w/o ) RTX overnight. The next day PBMCs were stained with a mixture of antibodies (anti-CD3 PE-Dazzle, anti-CD56 Bv421 and anti-CD16 FITC and CD69/CD137 PE) and analyzed by flow cytometry. NK cells were identified as CD3-CD56+ cells in the lymphocyte gate and fluorescence intensity of PE on CD56 dim NK cells was measured. a CD69 PE. Histogram shows one representative donor; gray incubation + RTX, black w/o RTX. Left diagram mean CD69 PE fluorescence intensity in samples from 13 donors. Statistical significance was determined with the Wilcoxon signed rank test (p = 0.0002). Right diagram Δ % in CD56 dim NK was determined by (CD69-positive CD56 dim NK cells after incubation + RTX) - (CD69-positive CD56 dim NK cells after incubation w/o RTX); bar median. b CD137(41BB)-PE; same donor, layout and abbreviations as in a ; n = 11 donors; p = 0.001

    Journal: Arthritis Research & Therapy

    Article Title: Rituximab induces phenotypical and functional changes of NK cells in a non-malignant experimental setting

    doi: 10.1186/s13075-016-1101-3

    Figure Lengend Snippet: Rituximab ( RTX ) leads to further phenotypical changes in CD56 dim natural killer ( NK ) cells. Freshly isolated peripheral blood mononuclear cells (PBMCs) were incubated with (+) or without ( w/o ) RTX overnight. The next day PBMCs were stained with a mixture of antibodies (anti-CD3 PE-Dazzle, anti-CD56 Bv421 and anti-CD16 FITC and CD69/CD137 PE) and analyzed by flow cytometry. NK cells were identified as CD3-CD56+ cells in the lymphocyte gate and fluorescence intensity of PE on CD56 dim NK cells was measured. a CD69 PE. Histogram shows one representative donor; gray incubation + RTX, black w/o RTX. Left diagram mean CD69 PE fluorescence intensity in samples from 13 donors. Statistical significance was determined with the Wilcoxon signed rank test (p = 0.0002). Right diagram Δ % in CD56 dim NK was determined by (CD69-positive CD56 dim NK cells after incubation + RTX) - (CD69-positive CD56 dim NK cells after incubation w/o RTX); bar median. b CD137(41BB)-PE; same donor, layout and abbreviations as in a ; n = 11 donors; p = 0.001

    Article Snippet: Flow cytometry PBMCs were stained for 20–30 minutes on ice with a cocktail of monoclonal antibodies, then washed and directly analyzed on a four-laser flow cytometer (LSR Fortessa, BD Biosciences, San Jose, CA, USA).

    Techniques: Isolation, Incubation, Staining, Flow Cytometry, Cytometry, Fluorescence

    Addition of rituximab to peripheral blood mononuclear cells ( PMBCs ) leads to B cell depletion in the absence of serum. a PBMCs were isolated by density gradient centrifugation, left untreated overnight, and cultured over the second night in medium supplemented with heat-inactivated fetal calf serum without any therapeutic antibody ( w/o , left panel ), with 10 μg/ml anti-CD20 antibody rituximab ( RTX ) ( middle ) or with 10 μg/ml anti-TNF alpha antibody infliximab ( right panel ). After that, PBMCs were stained with a mixture of lineage antibodies (anti-CD3 PE-Dazzle, anti-CD56 Bv421, anti-CD19 FITC) and analyzed by flow cytometry. B cells were identified as CD3-CD19+ cells in the lymphocyte gate. b PBMCs were treated as described ( a ). After washing PBMCs were re-diluted in Annexin-V (AxV) buffer supplemented with PE-labeled Annexin-V and analyzed by flow cytometry. B cells were identified as CD3-CD19+ cells in the lymphocyte gate. Upper row culture in medium without RTX ( w/o RTX ). Lower row culture in medium with RTX ( + RTX ). AxV+ B cells apoptotic B cells with positive staining for Annexin-V PE. a , b B cell numbers were reduced after incubation with RTX in 14/14 independent experiments, each performed with PBMCs from different healthy donors. In 10/14 experiments RTX-induced B cell depletion was complete as shown ( a ); in 4/14 B cell depletion was incomplete as shown ( b ). Infliximab was used as a negative control in 2/14 experiments. Increased binding of Annexin-V was identified in three donors with incomplete B cell depletion. c Left graph B cell percentages before and after culture overnight with rituximab (statistically significant, Wilcoxon signed rank test, p = 0.001). Right graph , whiskers 10–90th percentile. PBMCs with incomplete B cell depletion after incubation with RTX overnight ( incomplete ) had significantly lower ratios of natural killer cells to B cells ( NK/B ) than PBMCSs with complete B cell depletion ( complete ,

    Journal: Arthritis Research & Therapy

    Article Title: Rituximab induces phenotypical and functional changes of NK cells in a non-malignant experimental setting

    doi: 10.1186/s13075-016-1101-3

    Figure Lengend Snippet: Addition of rituximab to peripheral blood mononuclear cells ( PMBCs ) leads to B cell depletion in the absence of serum. a PBMCs were isolated by density gradient centrifugation, left untreated overnight, and cultured over the second night in medium supplemented with heat-inactivated fetal calf serum without any therapeutic antibody ( w/o , left panel ), with 10 μg/ml anti-CD20 antibody rituximab ( RTX ) ( middle ) or with 10 μg/ml anti-TNF alpha antibody infliximab ( right panel ). After that, PBMCs were stained with a mixture of lineage antibodies (anti-CD3 PE-Dazzle, anti-CD56 Bv421, anti-CD19 FITC) and analyzed by flow cytometry. B cells were identified as CD3-CD19+ cells in the lymphocyte gate. b PBMCs were treated as described ( a ). After washing PBMCs were re-diluted in Annexin-V (AxV) buffer supplemented with PE-labeled Annexin-V and analyzed by flow cytometry. B cells were identified as CD3-CD19+ cells in the lymphocyte gate. Upper row culture in medium without RTX ( w/o RTX ). Lower row culture in medium with RTX ( + RTX ). AxV+ B cells apoptotic B cells with positive staining for Annexin-V PE. a , b B cell numbers were reduced after incubation with RTX in 14/14 independent experiments, each performed with PBMCs from different healthy donors. In 10/14 experiments RTX-induced B cell depletion was complete as shown ( a ); in 4/14 B cell depletion was incomplete as shown ( b ). Infliximab was used as a negative control in 2/14 experiments. Increased binding of Annexin-V was identified in three donors with incomplete B cell depletion. c Left graph B cell percentages before and after culture overnight with rituximab (statistically significant, Wilcoxon signed rank test, p = 0.001). Right graph , whiskers 10–90th percentile. PBMCs with incomplete B cell depletion after incubation with RTX overnight ( incomplete ) had significantly lower ratios of natural killer cells to B cells ( NK/B ) than PBMCSs with complete B cell depletion ( complete ,

    Article Snippet: Flow cytometry PBMCs were stained for 20–30 minutes on ice with a cocktail of monoclonal antibodies, then washed and directly analyzed on a four-laser flow cytometer (LSR Fortessa, BD Biosciences, San Jose, CA, USA).

    Techniques: Isolation, Gradient Centrifugation, Cell Culture, Staining, Flow Cytometry, Cytometry, Labeling, Incubation, Negative Control, Binding Assay

    Rituximab ( RTX ) leads to natural killer ( NK ) cell degranulation and downregulation of CD16 in peripheral blood mononuclear cells (PBMCs). PBMCs were isolated and cultured as described in Fig. 1 . Anti-CD107a PE-Cy5 was added at the same time point as the therapeutic antibodies (starting point of the degranulation assay). The next day PBMCs were stained with a mixture of antibodies (anti-CD3 PE, anti-CD56 Bv421 and anti-CD16 FITC) and analyzed by flow cytometry. NK cells were identified as CD3-CD56+ cells in the lymphocyte gate. a CD107a expression on CD56 dim NK cells after stimulation without ( w/o ) therapeutic antibody, with 10 μg/ml RTX (+ RTX ), with 10 μg/ml infliximab and with 10 μg/ml intravenous immunoglobulin ( IvIg ) (from left to right ). Shown is one representative donor. b Summary of the increased CD107a expression on total NK cells after treatment with RTX in comparison to infliximab. Dots linked by a line belong to the same donor. Additive effect on degranulation is defined by (CD107a pos. NK cells after culture with therapeutic antibody) -(CD107a pos. NK cells after culture without therapeutic antibody). c CD16 expression on CD107a-positive NK cells. Shown is one representative donor. a - c CD107a expression together with CD16 expression has been investigated in healthy individuals ( n = 6 (+/− RTX), n = 3 (+/− infliximab) and n = 2 (+/− IvIg)). d The percentage of CD16 bright cells among CD56 dim NK cells before and after stimulation with RTX overnight was investigated in 16 healthy donors. Statistical significance was determined with the Wilcoxon signed rank test ( p = 0.0005). FSC forward scatter

    Journal: Arthritis Research & Therapy

    Article Title: Rituximab induces phenotypical and functional changes of NK cells in a non-malignant experimental setting

    doi: 10.1186/s13075-016-1101-3

    Figure Lengend Snippet: Rituximab ( RTX ) leads to natural killer ( NK ) cell degranulation and downregulation of CD16 in peripheral blood mononuclear cells (PBMCs). PBMCs were isolated and cultured as described in Fig. 1 . Anti-CD107a PE-Cy5 was added at the same time point as the therapeutic antibodies (starting point of the degranulation assay). The next day PBMCs were stained with a mixture of antibodies (anti-CD3 PE, anti-CD56 Bv421 and anti-CD16 FITC) and analyzed by flow cytometry. NK cells were identified as CD3-CD56+ cells in the lymphocyte gate. a CD107a expression on CD56 dim NK cells after stimulation without ( w/o ) therapeutic antibody, with 10 μg/ml RTX (+ RTX ), with 10 μg/ml infliximab and with 10 μg/ml intravenous immunoglobulin ( IvIg ) (from left to right ). Shown is one representative donor. b Summary of the increased CD107a expression on total NK cells after treatment with RTX in comparison to infliximab. Dots linked by a line belong to the same donor. Additive effect on degranulation is defined by (CD107a pos. NK cells after culture with therapeutic antibody) -(CD107a pos. NK cells after culture without therapeutic antibody). c CD16 expression on CD107a-positive NK cells. Shown is one representative donor. a - c CD107a expression together with CD16 expression has been investigated in healthy individuals ( n = 6 (+/− RTX), n = 3 (+/− infliximab) and n = 2 (+/− IvIg)). d The percentage of CD16 bright cells among CD56 dim NK cells before and after stimulation with RTX overnight was investigated in 16 healthy donors. Statistical significance was determined with the Wilcoxon signed rank test ( p = 0.0005). FSC forward scatter

    Article Snippet: Flow cytometry PBMCs were stained for 20–30 minutes on ice with a cocktail of monoclonal antibodies, then washed and directly analyzed on a four-laser flow cytometer (LSR Fortessa, BD Biosciences, San Jose, CA, USA).

    Techniques: Isolation, Cell Culture, Degranulation Assay, Staining, Flow Cytometry, Cytometry, Expressing

    The extent of rituximab-induced B cell depletion correlates with the size of natural killer ( NK ) cell proportions. NK cells were isolated from one fraction of freshly isolated peripheral blood mononuclear cells (PBMCs) and left untreated overnight. Another fraction of freshly isolated PBMCs was left untreated overnight and NK cells were depleted the next day using anti-CD56 and anti-CD16 monoclonal antibodies and magnetic beads. NK-cell-depleted PBMCs were resubstituted with isolated NK cells in three different proportions. The samples were incubated with ( gray bars ) or without ( black bars ) 10 μg/ml rituximab overnight. PBMCs were stained and analyzed by flow cytometry as described in Fig. 1 . a - c Three independent experiments. First bars show NK cell depletion without re-substitution. Second to fourth bars show NK cell depletion with resubstitution of different percentages of NK cells as indicated. a Data from the same experiment shown in Fig. 3 (cultured without human serum); the complete experiment with all negative controls including proof of successful NK cell depletion is shown in Additional file 1 : Figure S1(b). b , c Two further experiments performed with different donors (cultured with autologous human serum). The statistical analysis is described in “ Results ”

    Journal: Arthritis Research & Therapy

    Article Title: Rituximab induces phenotypical and functional changes of NK cells in a non-malignant experimental setting

    doi: 10.1186/s13075-016-1101-3

    Figure Lengend Snippet: The extent of rituximab-induced B cell depletion correlates with the size of natural killer ( NK ) cell proportions. NK cells were isolated from one fraction of freshly isolated peripheral blood mononuclear cells (PBMCs) and left untreated overnight. Another fraction of freshly isolated PBMCs was left untreated overnight and NK cells were depleted the next day using anti-CD56 and anti-CD16 monoclonal antibodies and magnetic beads. NK-cell-depleted PBMCs were resubstituted with isolated NK cells in three different proportions. The samples were incubated with ( gray bars ) or without ( black bars ) 10 μg/ml rituximab overnight. PBMCs were stained and analyzed by flow cytometry as described in Fig. 1 . a - c Three independent experiments. First bars show NK cell depletion without re-substitution. Second to fourth bars show NK cell depletion with resubstitution of different percentages of NK cells as indicated. a Data from the same experiment shown in Fig. 3 (cultured without human serum); the complete experiment with all negative controls including proof of successful NK cell depletion is shown in Additional file 1 : Figure S1(b). b , c Two further experiments performed with different donors (cultured with autologous human serum). The statistical analysis is described in “ Results ”

    Article Snippet: Flow cytometry PBMCs were stained for 20–30 minutes on ice with a cocktail of monoclonal antibodies, then washed and directly analyzed on a four-laser flow cytometer (LSR Fortessa, BD Biosciences, San Jose, CA, USA).

    Techniques: Isolation, Magnetic Beads, Incubation, Staining, Flow Cytometry, Cytometry, Cell Culture

    Suppression of CTL responses to the AAV capsid in vitro mediated by Tregitopes is contact mediated and results in anergy of effector cell . ( a ) Transwell experiment outline. Cells were placed in a transwell chamber in which the upper and lower chambers were separated by a permeable membrane (drawing). The table shows the different conditions tested in the transwell; in each of the transwell chambers untouched PBMC or PBMC depleted of CD4 + (CD4 neg CD8 + ) or CD8 + (CD4 + CD8 neg ) T cells were plated and restimulated with either AAV-only or AAV+hTreg167 peptides. After restimulation, PBMC or CD4 neg CD8 + cells were harvested from the transwell and used in a CTL assay against target cells loaded with the AAV-derived MHC I epitope VPYGYLTL at a Teff:target ratio 10:1. Results are expressed as % cytotoxicity compared to a max cytotoxicity (cells treated with 10% SDS) after background subtraction. ( b ) CTL assay in which target cells were transduced with an AAV vector at increasing MOIs and then incubated with HLA-matched effector cells. Teff cells were obtained by restimulating PBMC in vitro with the AAV capsid MHC I peptide VPQYGYLTL alone (AAV, black line), with AAV+Tregitope 167 (AAV+hTreg167, dashed black line), with AAV+hTreg167 followed by CD4 + T cell depletion (AAV+hTreg167 [CD4 neg ], gray line circles), or with AAV+hTreg167 followed by CD4 + T cell depletion and incubation with 10ng/ml IL-2 overnight (AAV+hTreg167 [CD4 neg + IL-2], gray line triangles). Teff:target ratio 10:1. Results are expressed as % cytotoxicity compared to a max cytotoxicity (cells treated with 10% SDS) after background subtraction. Experiments shown were repeated at least twice. Error bars represent SEM.

    Journal: Molecular Therapy

    Article Title: Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes

    doi: 10.1038/mt.2013.166

    Figure Lengend Snippet: Suppression of CTL responses to the AAV capsid in vitro mediated by Tregitopes is contact mediated and results in anergy of effector cell . ( a ) Transwell experiment outline. Cells were placed in a transwell chamber in which the upper and lower chambers were separated by a permeable membrane (drawing). The table shows the different conditions tested in the transwell; in each of the transwell chambers untouched PBMC or PBMC depleted of CD4 + (CD4 neg CD8 + ) or CD8 + (CD4 + CD8 neg ) T cells were plated and restimulated with either AAV-only or AAV+hTreg167 peptides. After restimulation, PBMC or CD4 neg CD8 + cells were harvested from the transwell and used in a CTL assay against target cells loaded with the AAV-derived MHC I epitope VPYGYLTL at a Teff:target ratio 10:1. Results are expressed as % cytotoxicity compared to a max cytotoxicity (cells treated with 10% SDS) after background subtraction. ( b ) CTL assay in which target cells were transduced with an AAV vector at increasing MOIs and then incubated with HLA-matched effector cells. Teff cells were obtained by restimulating PBMC in vitro with the AAV capsid MHC I peptide VPQYGYLTL alone (AAV, black line), with AAV+Tregitope 167 (AAV+hTreg167, dashed black line), with AAV+hTreg167 followed by CD4 + T cell depletion (AAV+hTreg167 [CD4 neg ], gray line circles), or with AAV+hTreg167 followed by CD4 + T cell depletion and incubation with 10ng/ml IL-2 overnight (AAV+hTreg167 [CD4 neg + IL-2], gray line triangles). Teff:target ratio 10:1. Results are expressed as % cytotoxicity compared to a max cytotoxicity (cells treated with 10% SDS) after background subtraction. Experiments shown were repeated at least twice. Error bars represent SEM.

    Article Snippet: The human hepatocyte cell line HHL5 (carrying the human HLA-A*0101 allele) and the HHL5 cell line genetically modified to express the human HLA-B*0702 allele, used as targets in the CTL assay, were previously described., Healthy donor PBMCs were obtained from Cellular Technologies (Cleveland, OH) and selected based on their HLA haplotype; so, they were carrier of the HLA-A*0101 or HLA-B*0702 alleles as well as matching the DRB1 alleles previously reported to be high-affinity binders of Tregitopes used in the study.

    Techniques: CTL Assay, In Vitro, Derivative Assay, Transduction, Plasmid Preparation, Incubation

    Antigen-specificity of Tregitopes-induced suppression of CTL responses is mediated by MHC I . ( a ) CTL assay in which target cells were loaded with the MHC I epitope VPQYGYLTL from AAV and incubated with HLA-matched AAV-specific Teff alone (AAV, dashed line), or Teff mixed at a 1:1 ratio with negatively-selected CD4 + T cells from AAV+hTreg167 restimulated PBMC (AAV+[AAV-hTreg167 CD4 + ], black line), or Teff mixed at a 1:1 ratio with negatively-selected CD4 + T cells from EBV+hTreg167 restimulated PBMC (AAV+[EBV-hTreg167 CD4 + ], gray line). ( b ) CTL assay as in ( a ) in which target cells were loaded with the MHC I epitope RPPIFIRRL from EBV and incubated with HLA-matched EBV-specific Teff alone (EBV, dashed line), or mixed 1:1 with negatively-isolated CD4 + T cells from EBV+hTreg167 cultures (EBV+[EBV-hTreg167 CD4 + ], gray line) or with negatively-isolated CD4 + T cells from AAV+hTreg167 cultures (EBV+[AAV-hTreg167 CD4 + ], black line). Teff:target ratio 10:1. Results are expressed as % cytotoxicity compared to a max cytotoxicity (cells treated with 10% SDS) after background subtraction. ( c ) MHC I blockade experiment. Black bars: PBMC were restimulated in vitro with AAV+hTreg167 and CD4 + T cells were negatively selected and incubated with increasing amounts of soluble AAV-specific TCR. After washing, CD4 + T cells were mixed 1:1 with AAV-specific Teff and added to targets. Grey bar: PBMC restimulated with AAV MHC I epitope only, used as positive control (AAV only). White bar: PBMC restimulated with AAV MHC I only mixed 1:1 with CD4 + T cells derived from cell restimulated with hTreg167 only, without AAV (hTreg167 only). AAV+AAV-TCR control, CTL in which AAV-only Teff were used the CTL assay in the presence of 20 µg/ml of AAV-TCR. Shown is the % cytotoxicity. Error bars represent the standard error of the mean of quadruplicate testing. Comparison of results with the AAV-only condition gave the following p values (unpaired, two-tailed t test): AAV-TCR 0 µg/ml P = 0.00262, 1 µg/ml P = 0.00016, 5 µg/ml P = 0.00112, 20 µg/ml P = 0.03335; hTreg167 only P = 0.86133; AAV+AAV-TCR P

    Journal: Molecular Therapy

    Article Title: Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes

    doi: 10.1038/mt.2013.166

    Figure Lengend Snippet: Antigen-specificity of Tregitopes-induced suppression of CTL responses is mediated by MHC I . ( a ) CTL assay in which target cells were loaded with the MHC I epitope VPQYGYLTL from AAV and incubated with HLA-matched AAV-specific Teff alone (AAV, dashed line), or Teff mixed at a 1:1 ratio with negatively-selected CD4 + T cells from AAV+hTreg167 restimulated PBMC (AAV+[AAV-hTreg167 CD4 + ], black line), or Teff mixed at a 1:1 ratio with negatively-selected CD4 + T cells from EBV+hTreg167 restimulated PBMC (AAV+[EBV-hTreg167 CD4 + ], gray line). ( b ) CTL assay as in ( a ) in which target cells were loaded with the MHC I epitope RPPIFIRRL from EBV and incubated with HLA-matched EBV-specific Teff alone (EBV, dashed line), or mixed 1:1 with negatively-isolated CD4 + T cells from EBV+hTreg167 cultures (EBV+[EBV-hTreg167 CD4 + ], gray line) or with negatively-isolated CD4 + T cells from AAV+hTreg167 cultures (EBV+[AAV-hTreg167 CD4 + ], black line). Teff:target ratio 10:1. Results are expressed as % cytotoxicity compared to a max cytotoxicity (cells treated with 10% SDS) after background subtraction. ( c ) MHC I blockade experiment. Black bars: PBMC were restimulated in vitro with AAV+hTreg167 and CD4 + T cells were negatively selected and incubated with increasing amounts of soluble AAV-specific TCR. After washing, CD4 + T cells were mixed 1:1 with AAV-specific Teff and added to targets. Grey bar: PBMC restimulated with AAV MHC I epitope only, used as positive control (AAV only). White bar: PBMC restimulated with AAV MHC I only mixed 1:1 with CD4 + T cells derived from cell restimulated with hTreg167 only, without AAV (hTreg167 only). AAV+AAV-TCR control, CTL in which AAV-only Teff were used the CTL assay in the presence of 20 µg/ml of AAV-TCR. Shown is the % cytotoxicity. Error bars represent the standard error of the mean of quadruplicate testing. Comparison of results with the AAV-only condition gave the following p values (unpaired, two-tailed t test): AAV-TCR 0 µg/ml P = 0.00262, 1 µg/ml P = 0.00016, 5 µg/ml P = 0.00112, 20 µg/ml P = 0.03335; hTreg167 only P = 0.86133; AAV+AAV-TCR P

    Article Snippet: The human hepatocyte cell line HHL5 (carrying the human HLA-A*0101 allele) and the HHL5 cell line genetically modified to express the human HLA-B*0702 allele, used as targets in the CTL assay, were previously described., Healthy donor PBMCs were obtained from Cellular Technologies (Cleveland, OH) and selected based on their HLA haplotype; so, they were carrier of the HLA-A*0101 or HLA-B*0702 alleles as well as matching the DRB1 alleles previously reported to be high-affinity binders of Tregitopes used in the study.

    Techniques: CTL Assay, Incubation, Isolation, In Vitro, Positive Control, Derivative Assay, Two Tailed Test

    Tregitopes can modulate CTL responses directed against several antigens and in the context of different HLA alleles . ( a–d ) Teff specific for Epstein-Barr virus (EBV), cytomegalovirus (CMV), hepatitis C virus (HCV), or influenza virus (Flu) were obtained following the protocol outlined in Figure S1. All MHC I epitopes were HLA-B*0702-restricted. Teff were used in the CTL assay at a Teff:target ratio of 1:10. Targets were peptide loaded with increasing concentrations of the relevant peptide. Results are expressed as % cytotoxicity compared to a max cytotoxicity (cells treated with 10% SDS) after background subtraction. Dashed line, Teff expanded from PBMC restimulated in vitro with indicated viral peptide only; Black line, Teff expanded in vitro with indicated viral peptide and hTreg167. ( e ) CTL assay in which Tregitope efficacy was tested against the AAV HLA-A*0101 restricted MHC I epitope SADNNNSEY. HLA-matched Teff were used in the CTL assay at a Teff:target ratio of 1:10. Targets were peptide loaded with increasing concentrations of the relevant peptide. Results are expressed as % cytotoxicity compared to a max cytotoxicity (cells treated with 10% SDS) after background subtraction. Dashed line, effectors obtained by restimulating PBMC in vitro with the SADNNNSEY epitope only; Black line, restimulation of PBMC was performed with the SADNNNSEY epitope from AAV and hTreg167. ( f ) CTL assay in which targets were loaded with the HLA-B*0702 epitope VPQYGYLTL from AAV. Teff were either obtained restimulating PBMC with the same AAV peptide only (AAV), or with the AAV peptide and Tregitope 167 (AAV+hTreg167), or with the AAV peptide and Tregitope 289 (AAV+hTreg289). Teff were used in the CTL assay at a Teff:target ratio of 1:10. Targets were peptide loaded with increasing concentrations of the relevant peptide. Results are expressed as % cytotoxicity compared to a max cytotoxicity (cells treated with 10% SDS) after background subtraction. Experiments shown were repeated at least twice. Error bars represent SEM.

    Journal: Molecular Therapy

    Article Title: Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes

    doi: 10.1038/mt.2013.166

    Figure Lengend Snippet: Tregitopes can modulate CTL responses directed against several antigens and in the context of different HLA alleles . ( a–d ) Teff specific for Epstein-Barr virus (EBV), cytomegalovirus (CMV), hepatitis C virus (HCV), or influenza virus (Flu) were obtained following the protocol outlined in Figure S1. All MHC I epitopes were HLA-B*0702-restricted. Teff were used in the CTL assay at a Teff:target ratio of 1:10. Targets were peptide loaded with increasing concentrations of the relevant peptide. Results are expressed as % cytotoxicity compared to a max cytotoxicity (cells treated with 10% SDS) after background subtraction. Dashed line, Teff expanded from PBMC restimulated in vitro with indicated viral peptide only; Black line, Teff expanded in vitro with indicated viral peptide and hTreg167. ( e ) CTL assay in which Tregitope efficacy was tested against the AAV HLA-A*0101 restricted MHC I epitope SADNNNSEY. HLA-matched Teff were used in the CTL assay at a Teff:target ratio of 1:10. Targets were peptide loaded with increasing concentrations of the relevant peptide. Results are expressed as % cytotoxicity compared to a max cytotoxicity (cells treated with 10% SDS) after background subtraction. Dashed line, effectors obtained by restimulating PBMC in vitro with the SADNNNSEY epitope only; Black line, restimulation of PBMC was performed with the SADNNNSEY epitope from AAV and hTreg167. ( f ) CTL assay in which targets were loaded with the HLA-B*0702 epitope VPQYGYLTL from AAV. Teff were either obtained restimulating PBMC with the same AAV peptide only (AAV), or with the AAV peptide and Tregitope 167 (AAV+hTreg167), or with the AAV peptide and Tregitope 289 (AAV+hTreg289). Teff were used in the CTL assay at a Teff:target ratio of 1:10. Targets were peptide loaded with increasing concentrations of the relevant peptide. Results are expressed as % cytotoxicity compared to a max cytotoxicity (cells treated with 10% SDS) after background subtraction. Experiments shown were repeated at least twice. Error bars represent SEM.

    Article Snippet: The human hepatocyte cell line HHL5 (carrying the human HLA-A*0101 allele) and the HHL5 cell line genetically modified to express the human HLA-B*0702 allele, used as targets in the CTL assay, were previously described., Healthy donor PBMCs were obtained from Cellular Technologies (Cleveland, OH) and selected based on their HLA haplotype; so, they were carrier of the HLA-A*0101 or HLA-B*0702 alleles as well as matching the DRB1 alleles previously reported to be high-affinity binders of Tregitopes used in the study.

    Techniques: CTL Assay, In Vitro

    In vitro restimulation of PBMC with Tregitopes results in the expansion of a population of suppressive CD4 + CD25 + T cells . Flow cytometry plots of PBMC restimulated in vitro with the AAV MHC I epitope VPQYGYLTL alone (AAV) ( a ) or with Tregitope 167 (AAV+hTreg167) ( b ). Cells were gated on lymphocytes after live/dead exclusion, CD3 + T cells, and CD4 + CD8 - and CD4 - CD8 + T cells were analyzed for CD25 + . ( c ) Histogram plot showing FoxP3 expression on CD4 + CD25 + T cells restimulated with AAV-only from (a) or AAV+hTreg167 from (b); the isotype control is shown in red. Mean fluorescent intensity (MFI) is indicated in the figure legend. ( d ) Suppression experiments in which CD4 + T cells (Tsupp) negatively isolated from PBMC restimulated in vitro with AAV+hTreg167 (blue line) or with AAV only (green line) were mixed at defined ratios with CD8 + Teff negatively isolated from PBMC restimulated in vitro with the AAV only. ( e ) CTL assay in which Teff were derived from PBMC restimulated in vitro with AAV alone (AAV) or with Tregitope 167 (AAV+hTreg167). Alternatively, PBMC were depleted of CD25 + T cells prior to AAV+hTreg167 restimulation (AAV+hTreg167 [CD25 neg ]). Teff:target ratio 10:1. Results are expressed as % cytotoxicity compared to a max cytotoxicity (cells treated with 10% SDS) after background subtraction. Experiments shown were repeated at least twice. Error bars represent SEM.

    Journal: Molecular Therapy

    Article Title: Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes

    doi: 10.1038/mt.2013.166

    Figure Lengend Snippet: In vitro restimulation of PBMC with Tregitopes results in the expansion of a population of suppressive CD4 + CD25 + T cells . Flow cytometry plots of PBMC restimulated in vitro with the AAV MHC I epitope VPQYGYLTL alone (AAV) ( a ) or with Tregitope 167 (AAV+hTreg167) ( b ). Cells were gated on lymphocytes after live/dead exclusion, CD3 + T cells, and CD4 + CD8 - and CD4 - CD8 + T cells were analyzed for CD25 + . ( c ) Histogram plot showing FoxP3 expression on CD4 + CD25 + T cells restimulated with AAV-only from (a) or AAV+hTreg167 from (b); the isotype control is shown in red. Mean fluorescent intensity (MFI) is indicated in the figure legend. ( d ) Suppression experiments in which CD4 + T cells (Tsupp) negatively isolated from PBMC restimulated in vitro with AAV+hTreg167 (blue line) or with AAV only (green line) were mixed at defined ratios with CD8 + Teff negatively isolated from PBMC restimulated in vitro with the AAV only. ( e ) CTL assay in which Teff were derived from PBMC restimulated in vitro with AAV alone (AAV) or with Tregitope 167 (AAV+hTreg167). Alternatively, PBMC were depleted of CD25 + T cells prior to AAV+hTreg167 restimulation (AAV+hTreg167 [CD25 neg ]). Teff:target ratio 10:1. Results are expressed as % cytotoxicity compared to a max cytotoxicity (cells treated with 10% SDS) after background subtraction. Experiments shown were repeated at least twice. Error bars represent SEM.

    Article Snippet: The human hepatocyte cell line HHL5 (carrying the human HLA-A*0101 allele) and the HHL5 cell line genetically modified to express the human HLA-B*0702 allele, used as targets in the CTL assay, were previously described., Healthy donor PBMCs were obtained from Cellular Technologies (Cleveland, OH) and selected based on their HLA haplotype; so, they were carrier of the HLA-A*0101 or HLA-B*0702 alleles as well as matching the DRB1 alleles previously reported to be high-affinity binders of Tregitopes used in the study.

    Techniques: In Vitro, Flow Cytometry, Cytometry, Expressing, Isolation, CTL Assay, Derivative Assay

    IgG-derived MHC class II epitopes (Tregitopes) mediate suppression of T cell responses in vitro . ( a ) CTL assay in which target cells were loaded with the AAV-derived MHC I epitope VPQYGYLTL (HLA-B*0702-restricted) at increasing concentrations and then incubated with HLA-matched Teff cells. Effectors were derived from HLA-B*0702 PBMC expanded in vitro against the same MHC I epitope alone (AAV, dashed line), or with hTreg167 (AAV+hTreg167, black line), or with three different scrambled versions of hTreg167 (Scramble 1, Scramble 2, and Scramble 3, grey lines). Teff:target ratio 10:1. Results are expressed as % cytotoxicity compared with a maximum cytotoxicity (cells treated with 10% SDS) after background subtraction. ( b ) CTL assay in which target cells were transduced with an AAV vector at increasing multiplicity of infections (MOIs) and then incubated with HLA-matched Teff cells restimulated with the AAV capsid MHC I peptide VPQYGYLTL alone (dashed line), or together with hTreg167 (black line). MOI, multiplicity of infection. Teff:target ratio 10:1. ( c ) Flow cytometry analysis of CD8 + T cell responses to the AAV MHC I epitope VPQYGYLTL. PBMC were restimulated in vitro in the presence of the AAV MHC I peptide VPQYGYLTL alone (AAV) or with hTreg167 (AAV+hTreg167) and then washed and incubated with the VPQYGYLTL peptide and stained for markers of T cell effector functions. Results are expressed as fold increase over non-restimulated PBMC incubated with the MHC I peptide VPQYGYLTL. For the analysis of flow cytometry data, after live/dead exclusion, cells were gated on lymphocytes, CD3 + T cells, and CD8 + T cells were then analyzed for IFN-γ, IL-2, TNF-α. SEB, Streptococcal enterotoxin B. Experiments shown were repeated at least twice. Error bars represent SEM. MFI, mean fluorescence intensity.

    Journal: Molecular Therapy

    Article Title: Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes

    doi: 10.1038/mt.2013.166

    Figure Lengend Snippet: IgG-derived MHC class II epitopes (Tregitopes) mediate suppression of T cell responses in vitro . ( a ) CTL assay in which target cells were loaded with the AAV-derived MHC I epitope VPQYGYLTL (HLA-B*0702-restricted) at increasing concentrations and then incubated with HLA-matched Teff cells. Effectors were derived from HLA-B*0702 PBMC expanded in vitro against the same MHC I epitope alone (AAV, dashed line), or with hTreg167 (AAV+hTreg167, black line), or with three different scrambled versions of hTreg167 (Scramble 1, Scramble 2, and Scramble 3, grey lines). Teff:target ratio 10:1. Results are expressed as % cytotoxicity compared with a maximum cytotoxicity (cells treated with 10% SDS) after background subtraction. ( b ) CTL assay in which target cells were transduced with an AAV vector at increasing multiplicity of infections (MOIs) and then incubated with HLA-matched Teff cells restimulated with the AAV capsid MHC I peptide VPQYGYLTL alone (dashed line), or together with hTreg167 (black line). MOI, multiplicity of infection. Teff:target ratio 10:1. ( c ) Flow cytometry analysis of CD8 + T cell responses to the AAV MHC I epitope VPQYGYLTL. PBMC were restimulated in vitro in the presence of the AAV MHC I peptide VPQYGYLTL alone (AAV) or with hTreg167 (AAV+hTreg167) and then washed and incubated with the VPQYGYLTL peptide and stained for markers of T cell effector functions. Results are expressed as fold increase over non-restimulated PBMC incubated with the MHC I peptide VPQYGYLTL. For the analysis of flow cytometry data, after live/dead exclusion, cells were gated on lymphocytes, CD3 + T cells, and CD8 + T cells were then analyzed for IFN-γ, IL-2, TNF-α. SEB, Streptococcal enterotoxin B. Experiments shown were repeated at least twice. Error bars represent SEM. MFI, mean fluorescence intensity.

    Article Snippet: The human hepatocyte cell line HHL5 (carrying the human HLA-A*0101 allele) and the HHL5 cell line genetically modified to express the human HLA-B*0702 allele, used as targets in the CTL assay, were previously described., Healthy donor PBMCs were obtained from Cellular Technologies (Cleveland, OH) and selected based on their HLA haplotype; so, they were carrier of the HLA-A*0101 or HLA-B*0702 alleles as well as matching the DRB1 alleles previously reported to be high-affinity binders of Tregitopes used in the study.

    Techniques: Derivative Assay, In Vitro, CTL Assay, Incubation, Transduction, Plasmid Preparation, Infection, Flow Cytometry, Cytometry, Staining, Fluorescence

    The IL-27 rs153109 A > G polymorphism enhanced pro-inflammatory cytokine release of the human peripheral blood mononuclear cells (PBMCs). The PBMCs of 50 healthy volunteers were incubated in vitro and stimulated with 500 ng/mL lipolysaccharide (LPS) for 8 h. Then the supernatant concentration of TNF-α, IL-6 and IL-1β in groups with different rs153109 genotypes were measured ( a - f ). The horizontal line indicates the mean expression level within each genotype group. The error bar represents standard error of the mean

    Journal: Critical Care

    Article Title: The interleukin-27 -964A > G polymorphism enhances sepsis-induced inflammatory responses and confers susceptibility to the development of sepsis

    doi: 10.1186/s13054-018-2180-0

    Figure Lengend Snippet: The IL-27 rs153109 A > G polymorphism enhanced pro-inflammatory cytokine release of the human peripheral blood mononuclear cells (PBMCs). The PBMCs of 50 healthy volunteers were incubated in vitro and stimulated with 500 ng/mL lipolysaccharide (LPS) for 8 h. Then the supernatant concentration of TNF-α, IL-6 and IL-1β in groups with different rs153109 genotypes were measured ( a - f ). The horizontal line indicates the mean expression level within each genotype group. The error bar represents standard error of the mean

    Article Snippet: Plasma collection, mononuclear cell isolation and LPS stimulation The peripheral blood mononuclear cells (PBMCs) were isolated from the patients with sepsis and healthy individuals by density gradient centrifugation with Lymphoprep™ (Axis-Shield PoCAS, Oslo, Norway).

    Techniques: Incubation, In Vitro, Concentration Assay, Expressing

    The IL-27 expressions in the peripheral blood mononuclear cells (PBMCs) isolated from 55 healthy volunteers. The IL-27 mRNA expression of PBMCs from 55 healthy individuals under lipopolysaccharide (LPS) stimulation (500 ng/mL) in groups with different rs153109 genotypes ( a , b ); the supernatant concentration of IL-27 under LPS stimulation in groups with different rs153109 genotypes ( c , d ). The horizontal line indicates the mean expression level in each genotype group. The error bar represents standard error of the mean

    Journal: Critical Care

    Article Title: The interleukin-27 -964A > G polymorphism enhances sepsis-induced inflammatory responses and confers susceptibility to the development of sepsis

    doi: 10.1186/s13054-018-2180-0

    Figure Lengend Snippet: The IL-27 expressions in the peripheral blood mononuclear cells (PBMCs) isolated from 55 healthy volunteers. The IL-27 mRNA expression of PBMCs from 55 healthy individuals under lipopolysaccharide (LPS) stimulation (500 ng/mL) in groups with different rs153109 genotypes ( a , b ); the supernatant concentration of IL-27 under LPS stimulation in groups with different rs153109 genotypes ( c , d ). The horizontal line indicates the mean expression level in each genotype group. The error bar represents standard error of the mean

    Article Snippet: Plasma collection, mononuclear cell isolation and LPS stimulation The peripheral blood mononuclear cells (PBMCs) were isolated from the patients with sepsis and healthy individuals by density gradient centrifugation with Lymphoprep™ (Axis-Shield PoCAS, Oslo, Norway).

    Techniques: Isolation, Expressing, Concentration Assay