Journal: Annals of the Rheumatic Diseases

Article Title: The STAT4 SLE risk allele rs7574865[T] is associated with increased IL-12-induced IFN-γ production in T cells from patients with SLE

doi: 10.1136/annrheumdis-2017-212794

Figure Lengend Snippet: Janus kinase inhibitors block the IL-12 and IFN-γ response in peripheral blood mononuclear cells (PBMCs) from STAT4 risk patients with systemic lupus erythematosus (SLE). (A) Healthy donor PBMCs were treated with serial dilutions of the JAK2 selective (JAK2i), the TYK2 selective (TYK2i) and the pan-JAK inhibitor (pan-JAKi) before being stimulated with 5 ng/mL IL-12, 0.1 ng/mL IFN-γ or 200 U/mL IFN-α for 20 min. IL-12-stimulated cells were preactivated with PHA/IL-2. Phosphorylation of STAT4 (pSTAT4) and STAT1 (pSTAT1) was determined by flow cytometry in IL-12-stimulated cells and IFN-γ or IFN-α-stimulated cells, respectively. Data (mean±SD of two individuals) from IL-12 and IFN-α-stimulated cells are shown for CD3 + T cells and from IFN-γ-stimulated cells for monocytes. (B) IC 50 values for each inhibitor. (C–E) PBMCs from healthy donors (HD), patients with SLE homozygous for the protective STAT4 allele (G/G) and patients with SLE carrying one or two STAT4 risk alleles (G/T+T/T) were incubated with 200 nM TYK2i (C, D), 70 nm JAK2i or 30 nM pan-JAKi (E). (C, D) Inhibition of IL-12-induced pSTAT4 in CD8 + T cells (C) and reduction in the frequency of IFN-γ + memory CD45RO + CD57 – CD8 + T cells (D). (E) Inhibition of IFN-γ-induced pSTAT1 in monocytes. (C–E) Open triangles and squares denote G/T and T/T patients with SLE, respectively.

Article Snippet: In some experiments, PBMCs were preactivated with 1.5% phytohaemagglutinin (PHA; Life Technologies) and 2.5 ng/mL IL-2 (Miltenyi) for 72 hours.

Techniques: Blocking Assay, Flow Cytometry, Cytometry, Incubation, Inhibition

Journal: Annals of the Rheumatic Diseases

Article Title: The STAT4 SLE risk allele rs7574865[T] is associated with increased IL-12-induced IFN-γ production in T cells from patients with SLE

doi: 10.1136/annrheumdis-2017-212794

Figure Lengend Snippet: PHA/IL-2 preactivated T cells from patients with systemic lupus erythematosus (SLE) carrying the STAT4 risk allele have an increased phosphorylation of STAT4 in response to IL-12. PHA/IL-2 pre-activated peripheral blood mononuclear cells were stimulated with 50 ng/mL IL-12 for 20 min. Phosphorylation of STAT4 (pSTAT4) was determined in indicated cell populations using flow cytometry. (A) Histograms from one representative donor. (B–D) Cumulative data from 19 homozygous protective (G/G, black circles), 20 heterozygous (G/T, open triangles) and 9 homozygous risk (T/T, open squares) STAT4 patients with SLE. Due to

Article Snippet: In some experiments, PBMCs were preactivated with 1.5% phytohaemagglutinin (PHA; Life Technologies) and 2.5 ng/mL IL-2 (Miltenyi) for 72 hours.

Techniques: Flow Cytometry, Cytometry

Journal: Annals of the Rheumatic Diseases

Article Title: The STAT4 SLE risk allele rs7574865[T] is associated with increased IL-12-induced IFN-γ production in T cells from patients with SLE

doi: 10.1136/annrheumdis-2017-212794

Figure Lengend Snippet: PHA/IL-2 preactivated T cells from STAT4 risk patients with systemic lupus erythematosus (SLE) have an increased IFN-γ production in response to IL-12. (A, B) PHA/IL-2 activated peripheral blood mononuclear cells were re-stimulated with 5 ng/mL IL-12 for 15 hours (A) or 20 ng/mL PMA together with 1 ng/mL A23187 for 6 hours (B). The frequency of IFN-γ + cells was determined in CD8 + and CD4 + T cells and subsets thereof using flow cytometry. Data from 18 STAT4 homozygous protective (G/G, black circles), 21 heterozygous (G/T, open triangles) and 9 homozygous risk (T/T, open squares) patients with SLE. Data of CD8 + T cells from one G/T individual (indicated as a filled triangle in A) was considered an outlier and was excluded from the statistical analysis of CD8 + T cells and subsets thereof. Due to

Article Snippet: In some experiments, PBMCs were preactivated with 1.5% phytohaemagglutinin (PHA; Life Technologies) and 2.5 ng/mL IL-2 (Miltenyi) for 72 hours.

Techniques: Flow Cytometry, Cytometry

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

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

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

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

Journal: Neuropsychopharmacology

Article Title: Selective DNA Methylation of BDNF Promoter in Bipolar Disorder: Differences Among Patients with BDI and BDII

doi: 10.1038/npp.2012.10

Figure Lengend Snippet: Levels of brain-derived neurotrophic factor (BDNF) mRNA in peripheral blood mononuclear cells from patients diagnosed with bipolar disorders type 1 (BD I; n =16) and BD type 2 (BD II; n =16). Box plots with whiskers from minimum to maximum represent 2 −DDCt

Article Snippet: Keywords: brain-derived neurotrophic factor (BDNF), peripheral blood mononuclear cells (PBMCs), DNA methylation, gene expression, bipolar disorder (BD), mood stabilizers and antidepressants

Techniques: Derivative Assay

Journal: Neuropsychopharmacology

Article Title: Selective DNA Methylation of BDNF Promoter in Bipolar Disorder: Differences Among Patients with BDI and BDII

doi: 10.1038/npp.2012.10

Figure Lengend Snippet: Amount of methylated DNA in the promoter region of brain-derived neurotrophic factor (BDNF) in peripheral blood mononuclear cells from patients diagnosed with bipolar disorders type 1 (BD I) or BD type 2 (BD II) in therapy with (+) or without

Article Snippet: Keywords: brain-derived neurotrophic factor (BDNF), peripheral blood mononuclear cells (PBMCs), DNA methylation, gene expression, bipolar disorder (BD), mood stabilizers and antidepressants

Techniques: Methylation, Derivative Assay

Journal: Neuropsychopharmacology

Article Title: Selective DNA Methylation of BDNF Promoter in Bipolar Disorder: Differences Among Patients with BDI and BDII

doi: 10.1038/npp.2012.10

Figure Lengend Snippet: Amount of methylated DNA in the promoter region of brain-derived neurotrophic factor (BDNF) in peripheral blood mononuclear cells from patients diagnosed with bipolar disorders type 1 (BD I) + BDs type 2 (BD II) in therapy with (+) or

Article Snippet: Keywords: brain-derived neurotrophic factor (BDNF), peripheral blood mononuclear cells (PBMCs), DNA methylation, gene expression, bipolar disorder (BD), mood stabilizers and antidepressants

Techniques: Methylation, Derivative Assay

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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