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: Cell Reports

Article Title: Hepatocytes Delete Regulatory T Cells by Enclysis, a CD4 + T Cell Engulfment Process

doi: 10.1016/j.celrep.2019.09.068

Figure Lengend Snippet: Anti-ICAM-1 Inhibits Early Adhesion and Enclysis of CD4 + T Cells Huh-7 cells (CMFDA, green) were co-cultured with lymphocytes. (A) Removing lymphocytes (CMPTX, red) by washes at the indicated times allowed measurement of early adhesion (5 min), firm binding (30 min), and trans-epithelial migration (60 min) by confocal microscopy. (B) Anti-ICAM-1 or isotype-matched control antibodies were added to a confluent Huh-7 monolayer before lymphocyte co-culture, and adhered lymphocytes (CMFDA, green) were washed away at the indicated times. Adhered lymphocytes were enumerated by total CMFDA fluorescence in replicate wells of a 96-well plate. (C) Huh-7 cells (CMFDA, green) were co-cultured with Jurkat T cells (BMQC, blue) for 3 h, fixed, and stained for ICAM-1 (anti-mouse Alexa 594, red) and then imaged by confocal microscopy. The scale bars represent 20 μm. (D) Huh-7 cells (CMFDA, green) were co-cultured with lymphocytes (CMTPX, red) in the presence of anti-ICAM-1, anti-ICAM-2, or isotype-matched control antibodies for 3 h and dislodged from plastic using EDTA. Huh-7 cells containing a lymphocyte were sorted by flow cytometry, and internalization was confirmed by confocal microscopy. Inhibition of lymphocyte internalization was enumerated compared with an isotype-matched control. The dotted line indicates assay variation cutoff across all conditions (±22%). Error bars indicate SD from at least three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; Student’s non-parametric paired t test (Wilcoxon). See also and .

Article Snippet: EasySep Human CD4 + CD127 low CD49d - kit was used for Treg isolation and a custom-made kit CD4 + CD25 - for non-Treg cells was also prepared by StemCell Technologies.

Techniques: Cell Culture, Binding Assay, Migration, Confocal Microscopy, Control, Co-Culture Assay, Fluorescence, Staining, Flow Cytometry, Inhibition

Journal: Cell Reports

Article Title: Hepatocytes Delete Regulatory T Cells by Enclysis, a CD4 + T Cell Engulfment Process

doi: 10.1016/j.celrep.2019.09.068

Figure Lengend Snippet: Enclysis Involves Membrane Blebbing and Is Distinct from Efferocytosis (A) Live, apoptotic (staurosporine-treated), or necrotic (heat-killed) T cells were co-cultured with Huh-7 cells for 3 h, fixed, and imaged by scanning electron microscopy. Images show primary CD4 + T cells isolated from peripheral blood. White arrows show membrane alterations on Huh-7 cells. (B) Kinetics profiles of live (blue), apoptotic (red), or necrotic (green) T cell capture, determined by time-lapse imaging, following co-culture with Huh-7 cells over 7 h and imaged every 15 min using a CQ1 high-content benchtop microscope. Cell-in-cell structures were enumerated in three fields of view from triplicate wells over time and plotted, with error bars showing SD. (C) Representative images showing capture of live, apoptotic, and necrotic CD4 + T cells (BMQC, red) by Huh-7 cells (CMFDA, green) at time (t) = 7 h.

Article Snippet: EasySep Human CD4 + CD127 low CD49d - kit was used for Treg isolation and a custom-made kit CD4 + CD25 - for non-Treg cells was also prepared by StemCell Technologies.

Techniques: Membrane, Cell Culture, Electron Microscopy, Isolation, Imaging, Co-Culture Assay, Microscopy

Journal: Cell Reports

Article Title: Hepatocytes Delete Regulatory T Cells by Enclysis, a CD4 + T Cell Engulfment Process

doi: 10.1016/j.celrep.2019.09.068

Figure Lengend Snippet: Enclysis Forms β-Catenin-Rich Vesicles and Is Distinct from Entosis and Suicidal Emperipolesis (A) Huh-7 cells (CMFDA, green) were co-cultured with Jurkat T cells (BMQC, blue), fixed, stained for anti-E-cadherin (anti-mouse Alexa Fluor 594, red), and imaged by confocal microscopy. The arrow indicates an E-cadherin + entotic vesicle containing another Huh-7 cell. (B) A binucleate Huh-7 cell containing a T cell in a vesicle devoid of E-cadherin staining. (C) Huh-7 cell containing a T cell in a β-catenin + enclytic vesicle. (D) Orthogonal confocal image of a 30-μm-thick section from formalin-fixed, paraffin-embedded tissue from a cirrhotic liver explant stained for β-catenin and the CD4 + T cell transcription factor Tbet. Immunohistochemistry reveals autofluorescence, which helps visualize hepatocyte cytoplasm (blue) and,typical for the liver, cell debris that autofluoresces in all channels (white). (E) Pre-treatment of Huh-7 cells with wortmannin, which inhibits suicidal emperipolesis, did not affect enclysis. However, the myosin-II and macropinocytosis inhibitor blebbistatin (50 μM) reduced T cell capture, as measured by confocal microscopy. (F) Entosis inhibitor treatment of Huh-7 cells with the ROCK inhibitors Y-27632 and H1152 (10 μM) did not affect enclysis. (G) Enclysis was perturbed by Huh-7 pre-treatment with the actin organization inhibitors latrunculin A (0.1 μM) and cytochalasin D (1 μM). Data were derived from three independent experiments. ∗∗ p < 0.01, ∗∗∗ p < 0.001, Student’s non-parametric paired t test (Wilcoxon). Scale bars represent 10 μm. See also .

Article Snippet: EasySep Human CD4 + CD127 low CD49d - kit was used for Treg isolation and a custom-made kit CD4 + CD25 - for non-Treg cells was also prepared by StemCell Technologies.

Techniques: Cell Culture, Staining, Confocal Microscopy, Formalin-fixed Paraffin-Embedded, Immunohistochemistry, Derivative Assay

Journal: Cell Reports

Article Title: Hepatocytes Delete Regulatory T Cells by Enclysis, a CD4 + T Cell Engulfment Process

doi: 10.1016/j.celrep.2019.09.068

Figure Lengend Snippet: Enclytic Vesicles Containing Live Stimulated CD4 + T Cells Did Not Acidify and Did Not Associate with LAMP-1 + Lysosomes (A) Huh-7 cells were co-cultured with necrotic (heat-killed) or live peripheral blood-derived CD4 + T cells activated via CD3+CD28/IL-2 (BMQC, blue) in the presence of pHrodo Red dextran in the culture medium (colorless, fluoresces red in acidic compartments). Complete internalization of T cells was confirmed by CellMask Plasma Membrane Deep Red addition to the medium, which labels all exposed cell membranes (white). Acidic endosomes arising from the membrane are seen in red in Huh-7 cells. The arrows indicate a phagosome containing a necrotic T cell and an enclytic vesicle containing a live T cell. (B) CD4 + T cells were isolated from peripheral blood and co-cultured overnight with Huh-7 cells in the presence of pHrodo Red dextran. Live (blue), apoptotic (staurosporine-treated, red), and necrotic (heat-killed, green) cells were imaged every 15 min on a CQ1 high-content benchtop microscope, and the proportion of pHrodo Red + vesicles was quantified at t = 0 (open bars) and t = 7.25 h (closed bars). Data shown are mean ± SD from triplicate wells (three fields of view) from one experiment. (C and D) Live, apoptotic (staurosporine-treated), or necrotic (heat-killed) lymphocytes were labeled (BMQC, blue) and co-cultured with Huh-7 cells (CMFDA, green) for 24 h. The cells were fixed and stained with anti-LAMP-1 (anti-mouse Alexa 594). Cell-in-cell structures (C) and LAMP-1 + vesicles (D) were enumerated by confocal microscopy. (E) IMARIS rendering of cross-sections of vesicles on Huh-7 cells (CMFDA, green) containing live or heat-killed T cells (BMQC, blue), with LAMP-1 + vesicles shown in red. Scale bars indicate 5 μm. See also Figure S7 .

Article Snippet: EasySep Human CD4 + CD127 low CD49d - kit was used for Treg isolation and a custom-made kit CD4 + CD25 - for non-Treg cells was also prepared by StemCell Technologies.

Techniques: Cell Culture, Derivative Assay, Clinical Proteomics, Membrane, Isolation, Microscopy, Labeling, Staining, Confocal Microscopy

Journal: Cell Reports

Article Title: Hepatocytes Delete Regulatory T Cells by Enclysis, a CD4 + T Cell Engulfment Process

doi: 10.1016/j.celrep.2019.09.068

Figure Lengend Snippet: Hepatocytes Captured Treg Cells More Efficiently than Non-Treg Cells and Selectively Acidified Treg Cell-Containing Vesicles (A) CD4 + T cells were isolated from peripheral blood (BMQC, blue) and co-cultured overnight with a confluent monolayer of Huh-7 cells (CMFDA, green). The cells were fixed and stained for Foxp3 to detect T regulatory (Treg) cells (red, anti-mouse immunoglobulin G [IgG]-Alexa 594). Approximately 60% of captured T cells were Foxp3 + . Foxp3 − T cells were labeled more brightly with BMQC and easy to distinguish. (B) Treg and non-Treg cells were isolated from peripheral blood using magnetic beads and added to wells containing Huh-7 cells at equal numbers. 3 h later, non-associating T cells were washed off, and internalized cells were enumerated by confocal microscopy. Each dot represents average internalized cells per 100 Huh-7 cells in independent experiments. (C) Treg and non-Treg cells were isolated from peripheral blood and co-cultured overnight with Huh-7 cells in the presence of colorless pHrodo Red dextran, which fluoresces under low-pH conditions. Bright acidic vesicles were enumerated in random fields of view and plotted for three independent experiments. Error bars show SD. ∗∗∗∗ p < 0.0001. Statistics were derived from an unpaired, two-tailed Student’s t test.

Article Snippet: EasySep Human CD4 + CD127 low CD49d - kit was used for Treg isolation and a custom-made kit CD4 + CD25 - for non-Treg cells was also prepared by StemCell Technologies.

Techniques: Isolation, Cell Culture, Staining, Labeling, Magnetic Beads, Confocal Microscopy, Derivative Assay, Two Tailed Test

Journal: Cell Reports

Article Title: Hepatocytes Delete Regulatory T Cells by Enclysis, a CD4 + T Cell Engulfment Process

doi: 10.1016/j.celrep.2019.09.068

Figure Lengend Snippet: Foxp3 + Cells Were Found in Hepatocytes More Frequently Than Tbet + Cells in Donor Livers and in Explants from Patients with Chronic Liver Disease (A) Immunohistochemistry images of Foxp3 + and Tbet + cells (DAB, brown) in livers from a patient with autoimmune hepatitis (AIH). (B and C) Absolute numbers (B) and frequencies (C) of Foxp3 + or Tbet + CD4 + T cells in hepatocytes in immunohistochemistry-stained sections from 80 patients with end-stage liver disease. Manual counts were performed by two independent scientists. PBC, primary biliary cholangitis; PSC, primary sclerosing cholangitis; ALD, alcoholic liver disease; NASH, non-alcoholic steatohepatitis; HBV, hepatitis B virus infection; HCV, hepatitis C virus infection. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, Student’s non-parametric paired t test (Wilcoxon).

Article Snippet: EasySep Human CD4 + CD127 low CD49d - kit was used for Treg isolation and a custom-made kit CD4 + CD25 - for non-Treg cells was also prepared by StemCell Technologies.

Techniques: Immunohistochemistry, Staining, Virus, Infection

Journal: Cell Reports

Article Title: Hepatocytes Delete Regulatory T Cells by Enclysis, a CD4 + T Cell Engulfment Process

doi: 10.1016/j.celrep.2019.09.068

Figure Lengend Snippet:

Article Snippet: EasySep Human CD4 + CD127 low CD49d - kit was used for Treg isolation and a custom-made kit CD4 + CD25 - for non-Treg cells was also prepared by StemCell Technologies.

Techniques: Plasmid Preparation, Activation Assay, Recombinant, Blocking Assay, Software