Journal: PLoS ONE

Article Title: A Flexible Model of HIV-1 Latency Permitting Evaluation of Many Primary CD4 T-Cell Reservoirs

doi: 10.1371/journal.pone.0030176

Figure Lengend Snippet: (A) Production of a primary cell model of HIV-1 latency. Total CD4 T cells were isolated from PBMC with a single-step negative-selection procedure with magnetic beads to remove unwanted cell subpopulations. Within 24 h, isolated cells were spinoculated at 1200× g for 2 h at room temperature with viral supernatants corresponding to NL4-3 GFP IRES Nef, NL4-3 Luciferase, or NL4-3 mCherry:Luciferase viruses as schematically depicted in (B). After spinoculation, cells were placed in medium containing 5 µM saquinavir and cultured for 3 days. Cells are then counted and plated in 96-well plates in medium containing 30 µM raltegravir and various stimulators. (C) Reactivation profiles of cells latently infected with NL4-3 GFP IRES Nef, NL4-3 Luciferase, or NL4-3 mCherry:Luciferase. Latently infected cells were cultured with medium alone or medium containing 200 nM PMA and 1.5 µM ionomycin and harvested after 24 or 48 hours of culture. GFP- or mCherry-expressing cells were quantified by flow cytometry, and the percentage of GFP + or mCherry cells was calculated based on uninfected controls. Luciferase levels are reported as relative light units (RLU) and have been normalized to total protein content in cell lysates to control for different cell proliferation rates. All samples were analyzed in triplicate with error bars representing +/− SD. Results are representative of those obtained in analyses of at least 10 independent donors with each virus. (D) Flow cytometric gating and analysis of cells latently infected with NL4-3 GFP IRES Nef or NL4-3 mCherry:Luciferase 24 or 48 hours after stimulation with PMA and ionomycin. Forward scatter versus side scatter plots show cells infected with virus and left unstimulated or stimulated for 24 h.

Article Snippet: Total CD4 T cells were isolated by negative selection, according to manufacturer's protocol, with the EasySep CD4+ T-cell Enrichment Kit (Stem Cell Technologies).

Techniques: Isolation, Selection, Magnetic Beads, Luciferase, Cell Culture, Infection, Expressing, Flow Cytometry, Control, Virus

Journal: PLoS ONE

Article Title: A Flexible Model of HIV-1 Latency Permitting Evaluation of Many Primary CD4 T-Cell Reservoirs

doi: 10.1371/journal.pone.0030176

Figure Lengend Snippet: (A) Cells were cultured in the presence (pre-treatment) or absence (no pre-treatment) of 30 µM raltegravir that was added immediately after spinoculation. After 3 days, cells were washed and stimulated with PMA+ionomycin in the presence or absence of 30 µM raltegravir (pre-activation). Results are representative of data obtained using three independent donors with each reporter virus. Error bars represent +/−SD of triplicate experiments. (B) CD4 T cells were isolated and pre-treated with 30 µM raltegravir, 250 nM AMD3100, 100 nM Efavirenz, or medium alone for 30 min before spinoculation. Cells were spinoculated and then cultured in the presence or absence of each antiretroviral drug. Three days after spinoculation, total DNA was isolated from the cells, and levels of HIV integration were determined by Alu-gag qPCR (left panel) or levels of total HIV DNA were determined by gag qPCR (right panel). Viral integration levels were compared in cultures incubated in medium alone versus in the presence of raltegravir (RTGR), AMD3100 (AMD), or Efavirenz (EFV) antiviral drugs to confirm the specificity of the assay. Data shown represent an average of six replicate PCR samples +/− SD. Data are presented as the number of copies of HIV DNA per 100 cells. (C) Three days after spinoculation, cells were either lysed for DNA isolation or stimulated with PMA+ionomycin for 24–72 hours. Peak GFP expression data are shown as the mean of three replicate samples. HIV integration was analyzed by Alu-gag qPCR to specifically detect integrated proviral DNA, and levels were normalized to levels obtained for the single copy RNaseP gene. Data shown are average of six replicate PCR samples +/− SD. Data are presented as copies of integrated HIV DNA/100 cells and the number of GFP+ cells/100 cells.

Article Snippet: Total CD4 T cells were isolated by negative selection, according to manufacturer's protocol, with the EasySep CD4+ T-cell Enrichment Kit (Stem Cell Technologies).

Techniques: Cell Culture, Activation Assay, Virus, Isolation, Incubation, DNA Extraction, Expressing

Journal: PLoS ONE

Article Title: A Flexible Model of HIV-1 Latency Permitting Evaluation of Many Primary CD4 T-Cell Reservoirs

doi: 10.1371/journal.pone.0030176

Figure Lengend Snippet: (A) CD4+CD45RO+ cells were purified by single step negative selection and HIV latency was established in these cells as described above. Cells were plated in 96-well plates and stimulated with 200 nM PMA with 1.5 µM ionomycin, anti-CD3+anti-CD28 beads (ratio 1∶1), 62.5 ng/ml IL-7, 10 µM prostratin, or left unstimulated for 24 h. Cells were stained with CD45RA-APC-H7, CCR7-PE-Cy7, CD27-APC, and CD45RO-FITC (NL4-3 mCherry:Luc) or CD45RO-PE (NL4-3 GFP), and analyzed for receptor expression and viral reporter expression. To obtain fold stimulation ratios, data were normalized as the percentage of cells expressing the viral reporter with the indicated stimulation divided by the % cells expressing the viral reporter in the absence of stimulation. Data shown represent an average of results obtained from four independent donors for each viral construct. Error bars represent +/− SEM. (B) CD4+CD45RO+ cells (upper panel) were sorted for CCR7+CD27+ central memory cells (T CM ) and CCR7-CD27+ transitional memory cells (T TM ). Cells were cultured for 2 days and then infected by spinoculation of NL4-3 mCherry:Luc. At the time of infection, cells were analyzed by flow cytometry for receptor expression to determine the relative levels of CCR7 expression in each sorted population (lower panel). (C) Latently infected cells were either left unstimulated or stimulated for 30 h with the indicated inducers. Two independent donors are shown, and fold change was determined as described above for luciferase levels.

Article Snippet: Total CD4 T cells were isolated by negative selection, according to manufacturer's protocol, with the EasySep CD4+ T-cell Enrichment Kit (Stem Cell Technologies).

Techniques: Purification, Selection, Staining, Expressing, Construct, Cell Culture, Infection, Flow Cytometry, Luciferase

Journal: EMBO Molecular Medicine

Article Title: Antioxidant nanozyme counteracts HIV‐1 by modulating intracellular redox potential

doi: 10.15252/emmm.202013314

Figure Lengend Snippet: A CEM‐GFP cells were pre‐treated with 50 ng/μl of Vs for 15 min and infected with 0.1 moi of CXCR4‐using HIV‐1 (NL‐4.3), and GFP fluorescence was measured at 488 nm as an indicator of HIV LTR activity. Vs treatment was repeated every 24 h for the experiment. B–D A similar assay was performed using Jurkat (CD4 + T‐cell line), and viral replication was assessed by (B) gag RT–PCR, (C) p24 ELISA in the culture supernatant, and (D) immunoblotting for p24 (viral capsid protein) in the whole cell lysate. E U937 (promonocytes) were pre‐treated with 50 ng/μl of Vs for 15 min followed by infection with 1 moi of CCR5 using HIV‐1 (NL‐AD8), and viral replication was measured by gag RT–qPCR at 24 h post‐infection (hpi). F Primary CD4 + T cells purified from human PBMCs (3 healthy donors) were activated, pre‐treated with 25 ng/μl Vs for 15 min, and infected with 0.05 moi of HIV‐1 NL‐4.3. Virus released in supernatant was quantified by p24 ELISA. Vs treatment was repeated every 48 h. Data information: All figures except (B) and (E) were analyzed by 2‐way ANOVA. (B), and (E) were analyzed by Mann–Whitney test. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05. Data are representative of results from three independent experiments performed in triplicate (mean ± SD). Source data are available online for this figure.

Article Snippet: Briefly, 50 × 10 6 PBMCs were thawed and CD4 + T cells were isolated using EasySep human CD4 + T‐cell isolation kit (Stem Cell Technologies, Canada).

Techniques: Infection, Fluorescence, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR, Purification, Virus, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Antioxidant nanozyme counteracts HIV‐1 by modulating intracellular redox potential

doi: 10.15252/emmm.202013314

Figure Lengend Snippet: Human monocyte‐derived macrophages (HMDMs) were pre‐treated with 12.5 ng/µl of Vs for 15 min, followed by infection with HIV‐1 NL‐AD8. Viral release in supernatant was quantified by p24 ELISA at 7 and 14 dpi. Vs treatment was repeated every 72 h. Data are obtained from one healthy donor in duplicate (mean ± SD). Survival of HIV‐infected primary CD4 + T cells was monitored by Annexin V/PI staining at 3 dpi in presence or absence of Vs treatment. Percentage of necrotic (PI + ), early apoptotic (Annexin V + ), and late apoptotic (Annexin V + /PI + ) cells were plotted . Data are aggregated from three healthy donors (mean ± SEM). Data information: *** P < 0.001, ** P < 0.01, ns—non‐significant analyzed by 2‐way ANOVA.

Article Snippet: Briefly, 50 × 10 6 PBMCs were thawed and CD4 + T cells were isolated using EasySep human CD4 + T‐cell isolation kit (Stem Cell Technologies, Canada).

Techniques: Derivative Assay, Infection, Enzyme-linked Immunosorbent Assay, Staining

Journal: EMBO Molecular Medicine

Article Title: Antioxidant nanozyme counteracts HIV‐1 by modulating intracellular redox potential

doi: 10.15252/emmm.202013314

Figure Lengend Snippet: Schematic representation of generation of expanded CD4 + T cells and reactivation. CD4 + T cells were sorted from PBMCs of ARV‐suppressed HIV‐infected individuals and expanded in presence of PHA, IL‐2, and autologous feeder PBMCs from healthy donor. Expanded CD4 + T cells from three patients were cultured in presence of IL‐2 and ARVs, with and without 25 ng/µl Vs for 21 days. Vs treatment was given for 15 min every 3 rd day. HIV transcripts were quantified by RT–qPCR at day 14, day 21, and at 24 h post‐stimulation of cells cultured for 21 days by prostratin. Limit of detection for RT–qPCR was 3 viral transcripts per million cells. At day 21, cells were stimulated with 1 µM prostratin for 24 h and HIV transcripts were quantified by RT–qPCR. Reduction in viral stimulation in Vs‐treated samples are represented as percentage values. ND—non‐determined. Aggregate plot for 3 patients from data (C). Total HIV‐1 DNA was determined up to 21 days in cells treated with ARVs or Vs + ARVs. Data Information: (B), (D), and (E) were analyzed by one‐way ANOVA with Tukey’s multiple correction. * P < 0.05, ns—non‐significant. Data are aggregated from three ARV‐suppressed HIV‐infected human subjects (mean ± SD).

Article Snippet: Briefly, 50 × 10 6 PBMCs were thawed and CD4 + T cells were isolated using EasySep human CD4 + T‐cell isolation kit (Stem Cell Technologies, Canada).

Techniques: Infection, Cell Culture, Quantitative RT-PCR

Journal: European journal of immunology

Article Title: Toll like Receptor 2 engagement on CD4 + T cells promotes TH9 differentiation and function

doi: 10.1002/eji.201646846

Figure Lengend Snippet: Transcriptional analysis of resting CD4+ T-cells co-stimulated via CD28 or TLR2. (A) Venn diagram representing genes expressed in response to anti-CD28− and/or TLR2− co-stimulation. (B) Top genes expressed in CD4+ T cells in response to antiCD28− or TLR2 co-stimulation. (C) Relative expression of Il9 gene after CD28− and/or TLR2 co-stimulation.

Article Snippet: Briefly, peripheral blood was obtained from healthy donors, and the T cells were isolated from the buffy coat using Ficoll-Hypaque (Amersham Biosciences) density gradient centrifugation, followed by isolation with EasySep Human Naïve CD4+ T cell isolation beads (STEM CELL Technology).

Techniques: Expressing

Journal: European journal of immunology

Article Title: Toll like Receptor 2 engagement on CD4 + T cells promotes TH9 differentiation and function

doi: 10.1002/eji.201646846

Figure Lengend Snippet: TLR2 engagement on CD4+ T cells enhances IL9 mRNA and protein expression driven by polyclonal activation and TGF-β and IL-4. Naïve CD4+T cells from WT mice were stimulated with anti-CD3 and anti-CD28 mAbs and exogenous TGF-β (5ng/ml) and IL-4 (10ng/ml), with or without P3CSK4 (1μg/ml) for 48h. (A) Relative expression of Il9 mRNA under polarizing conditions with or without P3CSK4 (n=3).

Article Snippet: Briefly, peripheral blood was obtained from healthy donors, and the T cells were isolated from the buffy coat using Ficoll-Hypaque (Amersham Biosciences) density gradient centrifugation, followed by isolation with EasySep Human Naïve CD4+ T cell isolation beads (STEM CELL Technology).

Techniques: Expressing, Activation Assay

Journal: European journal of immunology

Article Title: Toll like Receptor 2 engagement on CD4 + T cells promotes TH9 differentiation and function

doi: 10.1002/eji.201646846

Figure Lengend Snippet: TLR2, but no other TLR ligands increase TGF-β and IL-4 driven IL-9 cytokine secretion. Naive CD4+ T cells were activated with anti-CD3 and anti-CD28 mAbs in the presence of different TLR ligands (1μg/ml) under non-polarizing and TH9 polarizing conditions for 48 h. IL-9 (A) and IFN-γ (B) ELISA were measured by culture supernatants. Means ± SD of three independent experiments are shown. * p < 0.05, ** p < 0.01, NS denotes non-significant.

Article Snippet: Briefly, peripheral blood was obtained from healthy donors, and the T cells were isolated from the buffy coat using Ficoll-Hypaque (Amersham Biosciences) density gradient centrifugation, followed by isolation with EasySep Human Naïve CD4+ T cell isolation beads (STEM CELL Technology).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Toll like Receptor 2 engagement on CD4 + T cells promotes TH9 differentiation and function

doi: 10.1002/eji.201646846

Figure Lengend Snippet: TLR2 engagement on Antigen85B specific CD4+ T cells increases TH9 differentiation driven by TGF-β and IL-4. Naive Ag85B transgenic CD4+ T cells were co-incubated with TLR2 KO BMDM pulsed with Ag85B peptide (1μg/ml) and co-stimulated with or without TLR2 ligand (P3CSK4) under non-polarizing and TH9 polarizing conditions for 48 h. (A) CD4+ T cells were permeabilized and labeled with mAbs to IL-9 and IFN-γ and percentages of IL-9+ or IFN-γ+ cells were determined by flow cytometry. (B, C) IL-9 and IFN-γ were measured in culture supernatants by ELISA. Means ± SD of five independent experiments are shown. * p < 0.05, ** p < 0.01. NS denote non- significant.

Article Snippet: Briefly, peripheral blood was obtained from healthy donors, and the T cells were isolated from the buffy coat using Ficoll-Hypaque (Amersham Biosciences) density gradient centrifugation, followed by isolation with EasySep Human Naïve CD4+ T cell isolation beads (STEM CELL Technology).

Techniques: Transgenic Assay, Incubation, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Toll like Receptor 2 engagement on CD4 + T cells promotes TH9 differentiation and function

doi: 10.1002/eji.201646846

Figure Lengend Snippet: Effect of TLR2 engagement on the expression of TH9, TH1 and TH2 transcription factors under non-polarizing or TH9− polarizing conditions. Naïve CD4+ T cells were stimulated with anti-CD3 and anti-CD28 mAbs alone (non-polarizing) or combined with TGF-β and IL-4 (TH9− polarizing) and with or without P3CSK4. Batf (A), Pu.1 (B), Tbx21 (C) and Gata3 (D) mRNA expression was determined by RT-PCR and normalized to actin.

Article Snippet: Briefly, peripheral blood was obtained from healthy donors, and the T cells were isolated from the buffy coat using Ficoll-Hypaque (Amersham Biosciences) density gradient centrifugation, followed by isolation with EasySep Human Naïve CD4+ T cell isolation beads (STEM CELL Technology).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: European journal of immunology

Article Title: Toll like Receptor 2 engagement on CD4 + T cells promotes TH9 differentiation and function

doi: 10.1002/eji.201646846

Figure Lengend Snippet: TLR2 engagement on human CD4+ T cells enhances IL9 mRNA and protein expression driven by polyclonal activation and TGF-β and IL-4. Naïve CD4+ T cells from three human donors were stimulated with anti-CD3 (10ug/ml) and anti-CD28 mAbs (1ug/ml) and exogenous TGF-β (5ng/ml) and IL-4 (10ng/ml), with or without P3CSK4 (2μg/ml) for 48h. (A) Relative expression of Il9 mRNA under non-polarizing and polarizing conditions with or without P3CSK4 (n=3) is shown. (B) IL-9 cytokine in culture supernatants were determined by ELISA. Means ± SD of three technical replicates for each donor are shown.

Article Snippet: Briefly, peripheral blood was obtained from healthy donors, and the T cells were isolated from the buffy coat using Ficoll-Hypaque (Amersham Biosciences) density gradient centrifugation, followed by isolation with EasySep Human Naïve CD4+ T cell isolation beads (STEM CELL Technology).

Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Lenalidomide regulates CNS autoimmunity by promoting M2 macrophages polarization

doi: 10.1038/s41419-018-0290-x

Figure Lengend Snippet: a , b Flow cytometry analysis of Th1 and Th17 cells in spleen ( a ) and DLN ( b ) from vehicle- and lenalidomide-treated WT EAE mice at day 17. Representative fluorescence activated cell sorting (FACS) plots (left) and statistics from six mice per group (right) are shown; cells are gated for CD4 + T cells. c The total numbers of MNCs in whole spinal cord and brain were isolated from vehicle- and lenalidomide-treated mice on day 17 ( n = 6). d Flow cytometry analysis of Th1 and Th17 cells in CNS-infiltrating MNCs from vehicle- and lenalidomide-treated WT EAE mice at day 17. Representative FACS plots (left) and statistics from six mice per group (right) are shown; cells are gated for CD4 + T cells. Data are presented as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle-treated controls

Article Snippet: After 4 h, splenic CD4 + T cells were isolated from MOG-treated WT mice via magnetic separation (EasySep Mouse CD4 Isolation Kit, Stem Cell Technologies), labeled with1 μM CFSE (Biolegend) and subsequently cocultured with BMDMs at a ratio of 1:4 in RPMI 1640 (GIBCO) media supplemented with MOG 35–55 peptide (20 μg/ml) for 72 h. The proliferation of CD4 + T cells was assessed by flow cytometry based on CFSE dilution.

Techniques: Flow Cytometry, Fluorescence, FACS, Isolation

Journal: Cell Death & Disease

Article Title: Lenalidomide regulates CNS autoimmunity by promoting M2 macrophages polarization

doi: 10.1038/s41419-018-0290-x

Figure Lengend Snippet: a WT mice were immunized with MOG 35–55 and treated with lenalidomide (30 mg/kg, i.g.) or vehicle (0.9% CMC-Na, i.g.) combined with empty or clodronate liposome (50 mg/kg, i.v.) at indicated time points (bottom arrows represent lenalidomide or vehicle treatment, upper arrows represent liposome treatment). Mean clinical score is shown ( n = 15 per group). b , c Flow cytometry analysis of M2 and M1 macrophages in spleen ( b ) and DLN ( c ) from vehicle- and lenalidomide-treated WT EAE mice at day 17. Representative FACS plots (left) and statistics from six mice per group (right) are shown; cells are gated for F4/80 + cells. d Flow cytometry analysis of M2 macrophages in whole spinal cord and brain. CNS-infiltrating MNCs isolated from vehicle- and lenalidomide-treated WT EAE mice at day 17 stained for antibodies, including CD11b, CD206, and CD45. CD11b + CD45 hi CD206 + cells were M2 macrophages. Representative FACS plots (left) and statistics from six mice per group (right) are shown. e WT mice were immunized with MOG 35–55 on day 0 and 3 × 10 6 BMDMs treated with or without 25 nM lenalidomide for 4 h were injected intravenously into these mice on day 9, 14, and 19 ( n = 5 per group). f The mean clinical score of mice in e . g BMDMs were treated with or without 25 nM lenalidomide for 4 h. Splenic CD4 + T cells were isolated from MOG-treated WT mice, labeled with1 μM CFSE and subsequently cocultured with BMDMs at a ratio of 1:4 supplemented with MOG 35–55 peptide (20 μg/ml) for 72 h. The proliferation of CD4 + T cells was confirmed by flow cytometry analysis based on CFSE dilution. Left panel represents flowcytometric dot plot of CFSE-labeled CD4 + T cells, right panel shows the percentage of CD4 + T cells that have proliferated based on CFSE dilution ( n = 3 per group). Data are presented as means ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: After 4 h, splenic CD4 + T cells were isolated from MOG-treated WT mice via magnetic separation (EasySep Mouse CD4 Isolation Kit, Stem Cell Technologies), labeled with1 μM CFSE (Biolegend) and subsequently cocultured with BMDMs at a ratio of 1:4 in RPMI 1640 (GIBCO) media supplemented with MOG 35–55 peptide (20 μg/ml) for 72 h. The proliferation of CD4 + T cells was assessed by flow cytometry based on CFSE dilution.

Techniques: Flow Cytometry, Isolation, Staining, Injection, Labeling