Journal: Nature immunology

Article Title: The encephalitogenicity of T H 17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF

doi: 10.1038/ni.2031

Figure Lengend Snippet: (a) Naive CD4+CD25−CD62LhiCD44lo T cells from spleens of C57BL/6 mice were sorted by flow cytometry and activated with anti-CD3 and anti-CD28 in the presence of TGF-β plus IL-6, anti-IFN-γ and anti-IL-4 for 72 h (first stimulation). Cells were rested 2 days in the presence of IL-2 and then reactivated with anti-CD3 and anti-CD28 (second stimulation) during 72 h in the presence of TGF-β plus IL-6, TGF-β, IL-6 and IL-23, IL-23 or without added cytokines. Cells were then stimulated with PMA and ionomycin in the presence of GolgiPlug for the final 4 h, stained and analyzed by flow cytometry. CD4+ cells are shown. (b) Percentage of GM-CSF+ cells among CD4+IL-17A+ cells after the second stimulation. (c) GM-CSF, IL-10 and IL-17A concentrations in cell culture supernatants after the second stimulation measured by ELISA. (d) TH17 cells restimulated in the presence of TGF-β and IL-6 for 72 h were treated with anti-IL-10 or isotype control (goat IgG) and analyzed by flow cytometry. (e) GM-CSF concentrations in culture supernatants of cells stimulated as in (d) measured by ELISA. **p< 0.001. Data are representative of three independent experiments. (error bars, s.e.m).

Article Snippet: Anti-GM-CSF monoclonal antibody (clone 22E9.11; ATCC) was kindly provided by KaloBios Pharmaceuticals, Inc. (South San Francisco).

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

Journal: Nature immunology

Article Title: The encephalitogenicity of T H 17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF

doi: 10.1038/ni.2031

Figure Lengend Snippet: (a) Naive CD4+CD25−CD62LhiCD44lo T cells from spleens of C57BL/6 mice were FACS sorted and differentiated into TH17 cells during the first stimulation. Cells were then reactivated with anti-CD3 and anti-CD28 for 72 h in the presence of either IL-23 or TGF-β plus IL-6, in the presence of IL-1β (10 ng/ml) and/or TNF (10 ng/ml). CD4+ cells are shown. (b) GM-CSF, IL-17A, IL-21 and IL-22 concentrations in cell culture supernatants after the second stimulation measured by ELISA. (c) Naive CD4+ T cells from spleens of C57BL/6 and RORγt-deficient mice were activated during 72 h with anti-CD3 and anti-CD28 in the presence of either TGF-β and IL-6 or IL-1β, IL-6 and IL-23. Cells were then stimulated with PMA and ionomycin in the presence of GolgiPlug, stained and analyzed by flow cytometry. *p<0.01, **p < 0.001. Data are representative of two independent experiments. (error bars, s.e.m).

Article Snippet: Anti-GM-CSF monoclonal antibody (clone 22E9.11; ATCC) was kindly provided by KaloBios Pharmaceuticals, Inc. (South San Francisco).

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

Journal: Nature immunology

Article Title: The encephalitogenicity of T H 17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF

doi: 10.1038/ni.2031

Figure Lengend Snippet: (a) Splenocytes from MOG35-55-immuzed C57BL/6 mice were activated with MOG35-55 (20 μg/ml) in the presence of TGF-β plus IL-6, IL-23 or without added cytokines. Cells were then stimulated with PMA and ionomycin in the presence of GolgiPlug, stained and analyzed by flow cytometry. CD4+ cells are shown. (b) Percentage of GM-CSF+ cells among CD4+IL-17A+ cells after the second stimulation. (c) GM-CSF, IL-10, and IL-17A concentrations in cell culture supernatants measured by ELISA. (d) GM-CSF and IL-17A concentrations in supernatants of cell cultures stimulated as in (a) in the presence of TGF-β plus IL-6 and treated with anti-IL-10 or goat IgG. (e) GM-CSF and IL-17A concentrations in supernatants of cell cultures stimulated as in (a) without added cytokines and treated with anti-IL-23p19 or goat IgG (f) Clinical scores of irradiated wild-type recipient mice that received 5×106 CD4+ cells enriched from EAE splenocytes activated in the presence of IL-23. Mice were treated with either anti-GM-CSF or rat IgG from day 2 to day 35 post cell transfer. *p< 0.01; **p < 0.001. Data are representative of two (d, e and f) or four (a, b, and c) independent experiments (error bars, s.e.m).

Article Snippet: Anti-GM-CSF monoclonal antibody (clone 22E9.11; ATCC) was kindly provided by KaloBios Pharmaceuticals, Inc. (South San Francisco).

Techniques: Staining, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Irradiation

Journal: Nature immunology

Article Title: The encephalitogenicity of T H 17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF

doi: 10.1038/ni.2031

Figure Lengend Snippet: Splenocytes from wild-type- or Csf2−/−-MBPAc1-11 transgenic mice were activated with MBPAc1-11 peptide in the presence of IL-12 (TH1 conditions) or TGF-β plus IL-6, anti-IFN-γ and anti-IL-4 during 72 h (TH17 conditions). Cells were rested 2 days in the presence of IL-2 and then reactivated with MBPAc1-11 peptide in the presence of IL-12 (TH1 conditions) or IL-23 (TH17 conditions). After 72 h, cells were stimulated with PMA and ionomycin in the presence of GolgiPlug. (a) Flow cytometric analysis of IL-17A and IFN-γ expression in wild-type and Csf2−/− TH1 and TH17 cells after the second stimulation. CD4+ cells are shown. (b) IL-17A, IFN-γ and GM-CSF concentrations were measured by ELISA in the supernatants after the second stimulation. (c) RORγt and T-bet expression on gated CD4+IFN-γ+ (TH1) or CD4+IL-17A+ cells (TH17) analyzed by flow cytometry after the second stimulation. Filled histograms represent isotype controls. (d) Real time PCR analysis of Ahr, CCL20, CCR6, IL-23R and RORα expression in CD4+ T cells enriched by magnetic beads after the second stimulation from wild-type or Csf2−/− MBPAc1-11-splenocytes cultured in TH17 conditions. (e) Clinical scores of recipient mice that received 5×106 of either MBPAc1-11-specific wild-type or Csf2−/− TH1 or TH17 cells enriched by magnetic beads after the second stimulation. Pertussis toxin was injected i.p. on days 0 and 2 post transfer. *p< 0.01; **p < 0.001. Data are representative of two independent experiments. (error bars, s.e.m).

Article Snippet: Anti-GM-CSF monoclonal antibody (clone 22E9.11; ATCC) was kindly provided by KaloBios Pharmaceuticals, Inc. (South San Francisco).

Techniques: Transgenic Assay, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Real-time Polymerase Chain Reaction, Magnetic Beads, Cell Culture, Injection

Journal: Clinical and Vaccine Immunology : CVI

Article Title: Monofunctional and Polyfunctional CD8 + T Cell Responses to Human Herpesvirus 8 Lytic and Latency Proteins ▿ †

doi: 10.1128/CVI.00189-10

Figure Lengend Snippet: IFN-γ ELISPOT responses to four HHV-8 latency and lytic proteins. CD14− PBMC from healthy HLA A*0201-positive HHV-8-seropositive donors were stimulated with autologous, mature DC that were loaded with overlapping 15- to 20-mer peptides derived from each of the HHV-8 proteins LANA-1, K12, gB, and K8.1. IFN-γ production was measured by a DC-enhanced ELISPOT assay, and the number of spots produced by cells without peptide was subtracted from the number of spots produced by cells with peptide to give net values for spots. The donors (three or four donors used for each protein) are listed in each row, while the representative peptide numbers are listed in each column. The colors for the boxes represent the net number of IFN-γ spots per 106 cells as follows: white, <1 spot; light gray, 1 to 199 spots; dark gray, 200 to 399 spots; black, 400 to 599 spots; and red, ≥600 spots. Hot spots are circled for LANA-1 (A), K12 (B), gB (C), and K8.1 (D).

Article Snippet: CD14 + cells were isolated from donor peripheral blood mononuclear cells (PBMC) using anti-CD14 monoclonal antibody (MAb)-coated magnetic beads (Miltenyi Biotec, Auburn, CA).

Techniques: Enzyme-linked Immunospot, Derivative Assay, Produced

Journal: Clinical and Vaccine Immunology : CVI

Article Title: Monofunctional and Polyfunctional CD8 + T Cell Responses to Human Herpesvirus 8 Lytic and Latency Proteins ▿ †

doi: 10.1128/CVI.00189-10

Figure Lengend Snippet: IFN-γ ELISPOT responses to known epitopes and peptide families derived from the HHV-8 protein hot spots. CD14− PBMC from healthy HLA A*0201-positive HHV-8-seropositive donors were stimulated for 1 week with autologous mature DC that were loaded with peptide. IFN-γ production was measured as described for Fig. ​Fig.1.1. Mean + SE numbers of spot-forming cells from 10 experiments using three donors in response to DC loaded with peptide families are shown for LANA-1 (A) and for K12, gB, K8.1, and known gB492-500 (40) and K8.1209-217 (5) epitopes (B).

Article Snippet: CD14 + cells were isolated from donor peripheral blood mononuclear cells (PBMC) using anti-CD14 monoclonal antibody (MAb)-coated magnetic beads (Miltenyi Biotec, Auburn, CA).

Techniques: Enzyme-linked Immunospot, Derivative Assay

Journal: Clinical and Vaccine Immunology : CVI

Article Title: Monofunctional and Polyfunctional CD8 + T Cell Responses to Human Herpesvirus 8 Lytic and Latency Proteins ▿ †

doi: 10.1128/CVI.00189-10

Figure Lengend Snippet: Polyfunctional CD8+ T cell responses to known and novel HHV-8 epitopes. CD14− PBMC from healthy HLA A*0201-positive HHV-8-seropositive donors were stimulated for 1 week with autologous mature DC loaded with known HHV-8 epitopes (5, 6, 12, 40) (A) or novel epitopes from LANA-1 (B) or K12, gB, or K8.1 (C). Responses were measured by polychromatic flow cytometry, and net response values were averaged for three donors (mean + SE). Diagrams were generated using SPICE.

Article Snippet: CD14 + cells were isolated from donor peripheral blood mononuclear cells (PBMC) using anti-CD14 monoclonal antibody (MAb)-coated magnetic beads (Miltenyi Biotec, Auburn, CA).

Techniques: Flow Cytometry, Generated

Journal: Clinical and Vaccine Immunology : CVI

Article Title: Monofunctional and Polyfunctional CD8 + T Cell Responses to Human Herpesvirus 8 Lytic and Latency Proteins ▿ †

doi: 10.1128/CVI.00189-10

Figure Lengend Snippet: Polyfunctional CD8+ T cell responses to HHV-8 peptide families. CD14− PBMC from healthy HLA A*0201-positive HHV-8-seropositive donors were stimulated for 1 week with autologous mature DC loaded with peptide. Peptide families are shown for LANA-1 17 (A), K12 7 (B), gB 44 (C), and K8.1 21 (D). Responses were measured by polychromatic flow cytometry, and net response values were averaged for three donors (mean + SE). Diagrams were generated using SPICE.

Article Snippet: CD14 + cells were isolated from donor peripheral blood mononuclear cells (PBMC) using anti-CD14 monoclonal antibody (MAb)-coated magnetic beads (Miltenyi Biotec, Auburn, CA).

Techniques: Flow Cytometry, Generated

Journal: Journal of Translational Medicine

Article Title: Immune signatures in human PBMCs of idiotypic vaccine for HCV-related lymphoproliferative disorders

doi: 10.1186/1479-5876-8-18

Figure Lengend Snippet: 6-color flow cytometric analysis was performed on VK3-20 protein-stimulated monocytes, mDC/pDC cell populations and immunophenotype analysis of surface maturation/activation marker expression is shown . Values in each quadrant represent the percentage of positive cells.

Article Snippet: Cytokine production was assessed using the BD™ Cytometric Bead Array (CBA) tool (Becton Dickinson and Company), according to the instructions of the manufacturer.

Techniques: Activation Assay, Marker, Expressing

Journal: Immunity

Article Title: Role of MR1-driven signals and amphiregulin on the recruitment and repair function of MAIT cells during skin wound healing

doi: 10.1016/j.immuni.2022.12.004

Figure Lengend Snippet:

Article Snippet: Anti-FITC microbeads , Miltenyi , Cat# 130-048-701.

Techniques: Cytometry, Immunohistochemistry, Immunofluorescence, Purification, Control, Recombinant, Produced, BIA-KA, Red Blood Cell Lysis, CRISPR, Software, Imaging, Cream, Microscopy, Immunostaining

Journal: The EMBO Journal

Article Title: Secretion of mitochondrial DNA via exosomes promotes inflammation in Behçet's syndrome

doi: 10.15252/embj.2022112573

Figure Lengend Snippet: A–C mtDNA levels in the CS of HC‐derived leukocytes following PAMP/DAMP stimulation, with or without DNase I treatment. CD14 + monocytes (A), neutrophils (B), and lymphocytes (C) isolated from the peripheral blood of HCs were PAMP/DAMP stimulated utilizing: AL (ATP and LPS), NIG (nigericin and LPS), LPS, PMA, fMLP, CpG (ODN2006), cGAMP, or IM (ionomycin). The COXIII levels in purified DNA in CS were measured using qPCR after removing cell debris and DNase I treatment or mock digestion. D mtDNA levels in the CS of THP‐1 cells induced to undergo primary necrosis (1 st Nec), secondary necrosis (2 nd Nec), apoptosis (Apo), or pyroptosis (Pyro). After removal of cell debris, the supernatant was untreated (left, middle) or treated with Triton‐X100 (right), then undigested (left) or digested (middle, right) with DNase I. mtDNA levels were measured in purified DNA. E, F mtDNA levels in 16K‐ (E) and 100K‐EV (F) fractions in the CS of THP‐1 cells and CD14 + monocytes induced to undergo 1 st Nec, 2 nd Nec, Apo, or Pyro. 16K‐EVs and 100K‐EVs were separated using centrifugation at 16,000 g and 100,000 g , respectively. mtDNA levels in these EVs were measured by qPCR. G, H Secretion of mtDNA in 100K‐EVs by pyroptotic stimulation. THP1 cells were stimulated with LPS alone or LPS + ATP (G). HC–derived CD14 + monocytes were intracellularly stimulated with LPS enclosed in liposomes utilizing Avalanche‐Omni (H). mtDNA levels in 100K‐EVs were measured after 100K‐EVs were isolated from CS. I Deposition of dsDNA in 100K‐EVs. 100K‐EVs isolated from the CS of THP‐1 cells after LPS/ATP stimulation were fixed and stained with an anti‐dsDNA antibody, and then visualized with a secondary antibody conjugated to 10‐nm gold particles. Images were obtained using transmission immunoelectron microscopy. Black dots (white arrows) indicate the dsDNA deposition. Scale bar, 100 nm. Data information: Statistical analyses were performed using an ANOVA with Tukey's post‐hoc test (D–G) (mean ± SD; ** P < 0.01; NS, not statistically significant) and a Student's t ‐test (H) (mean ± SD; ** P < 0.01). For panel A‐C (mean ± SD). The data are representative of three independent experiments. Source data are available online for this figure.

Article Snippet: Human CD14 microbeads , Miltenyi Biotec , 130‐050‐201 , .

Techniques: Derivative Assay, Isolation, Purification, Centrifugation, Liposomes, Staining, Transmission Assay, Immuno-Electron Microscopy

Journal: The EMBO Journal

Article Title: Secretion of mitochondrial DNA via exosomes promotes inflammation in Behçet's syndrome

doi: 10.15252/embj.2022112573

Figure Lengend Snippet: A Leakage of mtDNA from the mitochondria into the cytosol. THP‐1 cells under normal, apoptotic, or pyroptotic conditions were stained with the PicoGreen (green) and MitoTracker‐Deep Red (red) dyes and visualized using confocal microscopy. Scale bar, 5 μm (left). Quantification of the number of PicoGreen‐positive dots in the cytosol (right). B Leaked cytosolic mtDNA levels following cell death induction. After removing the nuclei, the mitochondrial and cytosolic fractions from THP‐1 cells were isolated under normal, apoptotic, or pyroptotic conditions. DNA was purified from the cytosolic fraction, and the mtDNA levels were measured. C, D Cytosolic mtDNA levels in Casp1‐KO or Gsdmd‐KO cells undergoing pyroptosis. mtDNA and mitochondria in WT, Casp1‐KO, and Gsdmd‐KO‐THP‐1 cells induced to undergo pyroptosis were stained with the PicoGreen (green) and MitoTracker‐Deep Red (red) dyes and visualized using confocal microscopy. Scale bar, 5 μm (left). Quantification of the number of PicoGreen‐positive dots in the cytosol (right) (C). mtDNA level in the cytosolic fraction was measured by qPCR (D). E Mitochondria localization of NT‐gasdermin‐D. Ten minutes after THP1 cells were induced pyroptosis, mitochondria fraction was isolated and Western blotting was performed using anti‐gasdermin‐D antibody. F mtDNA level in 100K‐EVs. 100K‐EVs were isolated from CS of WT, Casp1‐KO, Gsdmd‐KO THP1 cells induced to undergo pyroptosis, and mtDNA level in 100K‐EVs was measured. G mtDNA levels in 100K‐EVs from pyroptotic cells with inhibited caspase‐1 or mROS. CD14 + monocytes were pre‐treated with Mito‐TEMPO or Ac‐YVAD‐cmk and then stimulated with LPS and ATP. The levels of mtDNA in 100K‐EVs in CS were measured. H mtDNA levels in 100K‐EVs from caspase‐1‐restored Casp1‐KO‐THP‐1 cells. The levels of mtDNA were measured in 100K‐EVs secreted by pyroptotic WT, Casp1‐KO, and FL‐Casp1‐Casp1‐KO‐THP‐1 cells. I mtDNA levels in 100K‐EVs from cells overexpressing aCasp1 or NT‐Gsdmd. aCasp1‐p10/p20 or NT‐Gsdmd were introduced into WT THP‐1 cells, and the extracellular mtDNA levels within 100K‐Evs were measured. Data information: Statistical analyses were performed using a Steel–Dwass test (A, C) (median; 25 th and 75 th percentiles; minimum and maximum value of a population; ** P < 0.01), and an ANOVA with Tukey's post‐hoc test (B, D, F–I) (mean ± SD; * P < 0.05, ** P < 0.01; NS, not statistically significant). The data are representative of two (E) and three (A–D, F–I) independent experiments. Source data are available online for this figure.

Article Snippet: Human CD14 microbeads , Miltenyi Biotec , 130‐050‐201 , .

Techniques: Staining, Confocal Microscopy, Isolation, Purification, Western Blot

Journal: The EMBO Journal

Article Title: Secretion of mitochondrial DNA via exosomes promotes inflammation in Behçet's syndrome

doi: 10.15252/embj.2022112573

Figure Lengend Snippet: A–C Increased inflammasome activation and pyroptosis in BS monocytes. HC ( n = 4)‐ and BS ( n = 6) ‐derived CD14 + monocytes were stimulated with LPS or ATP. The bioactivity of IL‐1β (Α) and the levels of LDH (B) in CS were measured. The levels of active caspase‐1 (p10) and mature IL‐1β (p17) in CS of HC ( n = 6)‐ and BS ( n = 6)‐CD14 + monocytes were evaluated through western blotting using anti‐Casp1 (p10) and anti–IL‐1β (p17) antibodies (left), respectively. The concentrations of caspase‐1 (p10) and IL‐1β (p17) in gel bands were determined using the ImageJ software, and statistical analyses were performed (right) (C). D Secretion levels of mtDNA via 100K‐EVs in BS monocytes. Neutrophils and CD14 + monocytes isolated from the peripheral blood of HC or BS patients were stimulated with nigericin or LPS. After DNA was purified from 100K‐EVs in the CS, the mtDNA levels were measured using qPCR. E Caspase‐1‐dependent over‐secretion of mtDNA in BS monocytes. HC and BS monocytes were pre‐treated with or without Ac‐YVAD‐cmk, and the cells were stimulated with LPS or ATP. The levels of mtDNA in 100K‐EVs in the CS were measured using qPCR. F Deposition of dsDNA and 8‐OHdG in MVBs of BS monocytes. After inducing pyroptosis via LPS and ATP, transmission immunoelectron microscopy was performed using an anti‐dsDNA (upper) antibody and an anti‐8‐OHdG (lower) antibody and a secondary antibody conjugated to 10‐nm gold particles. Black dots (white arrows) indicate dsDNA‐ (left) or 8‐OHdG‐ (right) positive depositions. Scale bar, 1 μm. G–I Increased secretion of mtDNA in 100K‐EVs by an NLRP3‐mutant derivative. Full‐length NLRP3 (WT‐NLRP3) and an NLRP3 mutant (V200M‐NLRP3) were restored into NLRP3‐KO (FL‐NLRP3‐NLRP3‐KO and V200M‐NLRP3‐NLRP3‐KO) THP‐1 cells (G). The cells were subsequently stimulated with LPS and ATP. The levels of mtDNA in 100K‐EVs (H) and IL‐1β in the CS (I) were measured. Data information: Statistical analyses were performed using an ANOVA with Tukey's post‐hoc test (A, C–E, H, I) (mean ± SD; * P < 0.05, ** P < 0.01; NS, not statistically significant) and a Mann–Whitney U test (B) (median; 25 th and 75 th percentile; minimum and maximum value of a population; ** P < 0.01). The data are representative of two (A–F) and three (G–I) independent experiments. Source data are available online for this figure.

Article Snippet: Human CD14 microbeads , Miltenyi Biotec , 130‐050‐201 , .

Techniques: Activation Assay, Derivative Assay, Western Blot, Software, Isolation, Purification, Transmission Assay, Immuno-Electron Microscopy, Mutagenesis, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Secretion of mitochondrial DNA via exosomes promotes inflammation in Behçet's syndrome

doi: 10.15252/embj.2022112573

Figure Lengend Snippet: mROS production in CD14 + monocytes. HC and BS monocytes were stained with MitoPB (green) and MitoSox (red), and mROS production was visualized using confocal microscopy. Scale bar, 5 μm. Mitochondrial membrane potential in CD14 + monocytes. HC‐ and BS‐derived CD14 + monocytes were stimulated with LPS, and the reduction in the MMP was monitored by FACS using MitoTracker Green (X‐axis) and MitoTracker‐Deep Red (Y‐axis) (left). The increase in the proportion of MMP‐reduced cells (gated population) following LPS stimulation was quantified (right). Effect of caspase‐1 on the increasing MMP of BS monocytes. BS‐derived CD14 + monocytes were stimulated with LPS in the presence or absence of Ac‐YVAD‐cmk, and the reduction in the MMP was monitored using FACS. Data information: Statistical analyses were performed using a Mann–Whitney U test (B, C) (median; 25 th and 75 th percentiles; minimum and maximum value excluding outliers; * P < 0.05). The data are representative of two (A) and three (B, C) independent experiments. Source data are available online for this figure.

Article Snippet: Human CD14 microbeads , Miltenyi Biotec , 130‐050‐201 , .

Techniques: Staining, Confocal Microscopy, Membrane, Derivative Assay, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Secretion of mitochondrial DNA via exosomes promotes inflammation in Behçet's syndrome

doi: 10.15252/embj.2022112573

Figure Lengend Snippet:

Article Snippet: Human CD14 microbeads , Miltenyi Biotec , 130‐050‐201 , .

Techniques: Western Blot, Purification, Produced, Affinity Purification, Flow Cytometry, Confocal Microscopy, Electron Microscopy, Amplification, Recombinant, Blocking Assay, Adjuvant, Enzyme-linked Immunosorbent Assay, LDH Cytotoxicity Assay, Isolation, DNA Extraction, dsDNA Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation, Cloning, Mutagenesis, Sequencing, Transfection, DNA Ligation, Fractionation

Journal: Molecular cell

Article Title: MRI is a DNA Damage Response Adaptor during Classical Non-Homologous End Joining

doi: 10.1016/j.molcel.2018.06.018

Figure Lengend Snippet: (A) Number of live births produced from intercrossing MRI−/−:XLF+/− mice. (B) MRI−/− and MRI−/−:XLF−/− littermates at day E16.5. Scale bar, 1 cm. (C) Micrographs of the ganglionic eminence (GE, top row) and cortex (bottom row) in MRI−/−:XLF+/− and MRI−/−:XLF−/− embryos at days E14.5 and E16.5, respectively, stained with DAPI (blue) and anti-cleaved caspase 3 antibody (red). Scale bar, 100 m. See also Tables S1 and S2.

Article Snippet: Sections were blocked for 30 minutes, followed by incubation with primary rabbit anti-cleaved caspase 3 (1:200, Cell Signaling) overnight at 4°C.

Techniques: Produced, Staining

Journal: Molecular cell

Article Title: MRI is a DNA Damage Response Adaptor during Classical Non-Homologous End Joining

doi: 10.1016/j.molcel.2018.06.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sections were blocked for 30 minutes, followed by incubation with primary rabbit anti-cleaved caspase 3 (1:200, Cell Signaling) overnight at 4°C.

Techniques: Blocking Assay, Recombinant, Cell Isolation, Magnetic Beads, Mass Spectrometry, Magnetic Resonance Imaging, Sequencing, Plasmid Preparation, Software, High Content Screening, Flow Cytometry, Inverted Microscopy, Laser-Scanning Microscopy, Spectrophotometry, Irradiation

Journal: Cell

Article Title: HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis

doi: 10.1016/j.cell.2020.09.054

Figure Lengend Snippet: The Expression Level of DR2a Is Higher Than that of DR2b on Blood B Cells and Monocytes, Related to Figure 1 and (A) Workflow diagram for the generation of DR2a (mIgG1 isotype) and DR2b (mIgG2b isotype) allele-specific monoclonal antibodies. (B) Specificity of the DR2a and DR2b allele-specific monoclonal antibodies was examined with LABScreen™ system that uses microbeads coated with purified Class II HLA antigens and pre-optimized reagents for the detection of Class II HLA antibodies using flow cytometric technology. (C) Expression levels of DR2a and DR2b on blood B cells and monocytes of HLA-DR15 + HDs (n = 3) and MS patients (RRMS, n = 3; RRMS_NAT, n = 3) were detected by flow cytometry using Alexa Fluor 488-conjugated DR2a-specific antibody and Alexa Fluor 647-conjugated DR2b-specific antibody. (D) Normalized expression levels of DR2a and DR2b on blood B cells (left) and monocytes (right) from HLA-DR15 + HDs and MS patients were analyzed by flow cytometry using Alexa Fluor 488-conjugated DR2a-specific antibody and Alexa Fluor 647-conjugated DR2b-specific antibody. The PE-conjugated monomorphic antibody L243, which binds to the HLA-DR alpha chain and thus recognizes both DR2a and DR2b, was used to normalize the signals obtained with the two allele-specific antibodies. In brief, for the normalization, the ratios of the mean fluorescence intensity (MFI) of the allele-specific antibody versus the MFI of L243 on BLS-DR2a or BLS-DR2b cells were determined for each measurement. The resulting ratio was used to correct the MFI value of DR2a and DR2b expression on primary B cells and monocytes. Data are expressed as mean ± SEM.

Article Snippet: CD4 MicroBeads, human , Miltenyi Biotec , Cat# 130-045-101.

Techniques: Expressing, Bioprocessing, Purification, Flow Cytometry, Fluorescence

Journal: Cell

Article Title: HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis

doi: 10.1016/j.cell.2020.09.054

Figure Lengend Snippet: HLA-DR-SPs Mainly Activate Memory CD4 + T Cells in HLA-DR15 + MS Patients, Related to and and (A) Correlation of the total number of HLA-DR-SPs presented by the two HLA-DR15 molecules on B cells and the degree of autoproliferation in MS patients (RRMS, n = 3; RRMS_NAT, n = 3; Spearman’s rank correlation test). (B) PBMCs were separated into CD45RA + cells and CD45RA − cells by magnetic cell isolation using CD45RA microbeads. Cell subsets in untouched PBMCs, CD45RA + PBMCs, and CD45RA − PBMCs were analyzed by flow cytometry. (C) Comparison of the autoproliferation between untouched PBMCs and CD45RA − PBMCs from HLA-DR15 + HDs (n = 6), RRMS (n = 4), or RRMS_NAT (n = 6). Data are expressed as mean, and p values were determined by paired t test. (D) CD45RA − PBMCs from HLA-DR15 + HDs (n = 5) and MS patients (n = 5) were stimulated with Tetanus Toxin peptides, TT (830-844) and TT (947-967) , which could be presented by DR2a and/or DR2b as previously reported. For each sample, each stimulation was performed with 10 replicate wells. Proliferations of CD45RA − PBMCs were detected after 7 days by 3 H-thymidine incorporation assay, and the proliferation strength is depicted as SI. The p values were determined by unpaired t test. (E) CD45RA − PBMCs from HLA-DR15 + RRMS (n = 4) and RRMS_NAT (n = 10) patients were stimulated with the most common HLA-DR-SPs either alone or as pool. Proliferations of CD45RA − PBMCs were detected by 3 H-thymidine incorporation assay after 7 days, and proliferation strength is depicted as counts per minute (cpm). 10-15 replicate wells per condition are indicated by individual dots. The blue dotted line indicates the mean value of the no peptide control. The purple dotted line indicates the mean value plus three standard deviations of the no peptide control. Values above the purple dotted line were considered positive.

Article Snippet: CD4 MicroBeads, human , Miltenyi Biotec , Cat# 130-045-101.

Techniques: Cell Isolation, Flow Cytometry, Comparison, Thymidine Incorporation Assay, Control

Journal: Cell

Article Title: HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis

doi: 10.1016/j.cell.2020.09.054

Figure Lengend Snippet: Increased Reactivity of Memory CD4 + T Cells against HLA-DR-SPs in MS Patients (A) Responses of CD45RA − PBMCs of HLA-DR15 + HDs (n = 8) and MS patients (RRMS, n = 4; RRMS_NAT, n = 10) against the five most common HLA-DR-SPs alone or as pool. 10–15 replicate wells were tested for proliferation, and responses are depicted as the stimulatory index (SI). Individual wells are represented by dots for HDs (left) or MS patients (right). The purple dotted line indicates SI = 2, and SI ≥ 2 was considered positive. Responses to control CEF II peptides are shown at the bottom. (B) Proliferations of CD45RA − PBMCs from HDs and MS patients after stimulation with pooled HLA-DR-SPs in the presence of a blocking anti-HLA-DR Ab. (C) CD45RA − PBMCs from HLA-DR15 + MS patients were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with pooled HLA-DR-SPs. After 7 days, cells were analyzed, and proportions of memory CD4 + T cells in the proliferating (CFSE dim ) and highly proliferating (CFSE low ) compartments are shown in the pie charts. (D) Blood CD4 + T cells and monocytes were purified from PBMCs of HLA-DR15 + MS patients and co-cultured with pooled HLA-DR-SPs for 7 days. Proliferation of naive CD4 + T cells (CD45RA + ) and memory CD4 + T cells (CD45RA − ) was detected. (E) Th1/Th2/Th17-related cytokines in supernatants of CD45RA − PBMCs after stimulation with pooled HLA-DR-SPs. (F) IFN-γ, IL-17A, and IL-17F in supernatants of co-cultured blood CD4 + T cells and monocytes after stimulation with pooled HLA-DR-SPs. (G) Expression levels of CCR6 and CXCR3 on non-proliferating (CFSE high ) and proliferating (CFSE dim ) memory CD4 + T cells after stimulation with pooled HLA-DR-SPs for 7 days. Data are expressed as mean ± SEM, and p values were determined by unpaired t test. See also Figure S4 and and .

Article Snippet: CD4 MicroBeads, human , Miltenyi Biotec , Cat# 130-045-101.

Techniques: Control, Blocking Assay, Labeling, Purification, Cell Culture, Expressing

Journal: Cell

Article Title: HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis

doi: 10.1016/j.cell.2020.09.054

Figure Lengend Snippet: Responses of Autoreactive CD4 + TCCs to RASGRP2 and HLA-DR-SPs (A) Dose-response curves of TCC14 to RASGRP2 (78–87) and HLA-DR-SPs. (B) Irradiated BLS-DR2b cells were incubated with peptides for 12 h and then co-cultured with TCC14. Shown are expression levels of CD69 and CD25 on TCC14 at different time points after co-culture. ΔMFI indicates the mean fluorescence intensity (MFI) value above the no-peptide control. (C–E) CD45RA − PBMCs from 3 HLA-DR15 + RRMS_NAT patients were stimulated with RASGRP2 (78–87) to generate TCCs. Proliferation of memory CD4 + T cells was analyzed on day 12 (C). Acquired TCCs were co-cultured with irradiated autologous PBMCs as APCs and stimulated with RASGRP2 (78–87) or DRB1 (57–70) , and proliferation was detected on day 3. The TCCs responding to RASGRP2 (78–87) and DRB1 (57–70) are highlighted in blue (D). Five new 1159AG_TCCs that responded to RASGRP2 (78–87) and DRB1 (57–70) were generated from RRMS_NAT-2. Their corresponding TCRVβ sequence and functional phenotype are shown (E). (F) Restriction of the RASGRP2 (78–87) -specific 1159AG_TCCs was tested with irradiated BLS-DR2b cells and stimulation with RASGRP2 (78–87) . (G) Th1/Th2/Th17-related cytokines in supernatants of 1159AG_TCCs after co-culture with irradiated BLS-DR2b cells and stimulation with RASGRP2 (78–87) for 3 days. (H and I) 1159AG_TCCs were co-cultured with irradiated BLS-DR2b cells and stimulated with RASGRP2 (78–87) or DRB1/5 (184–199) for 3 days. Shown is proliferation of 1159AG_TCCs (H) and IFN-γ in supernatants (I). Data are expressed as mean ± SEM, and p values were determined by unpaired t test. See also and and .

Article Snippet: CD4 MicroBeads, human , Miltenyi Biotec , Cat# 130-045-101.

Techniques: Irradiation, Incubation, Cell Culture, Expressing, Co-Culture Assay, Fluorescence, Control, Generated, Sequencing, Functional Assay

Journal: Cell

Article Title: HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis

doi: 10.1016/j.cell.2020.09.054

Figure Lengend Snippet: DR2a and DR2b Present HLA-DR-SPs to Autoreactive CD4 + T Cells (A and B) Purified blood CD4 + T cells of HLA-DR15 + MS patients were co-cultured with irradiated BLS-DR2a or BLS-DR2b cells as APCs and stimulated with pooled HLA-DR-SPs for 7 days. Shown is proliferation of naive (CD45RA + ) and memory (CD45RA − ) CD4 + T cells (A) and IFN-γ in supernatants (B). (C) CSF-infiltrating CD4 + T cells of HLA-DR15 + MS patients (n = 9) were co-cultured with irradiated BLS-DR2a or BLS-DR2b cells and stimulated with pooled HLA-DR-SPs for 7 days. Proliferation of CSF-infiltrating CD4 + T cells is shown. The bar graph at the bottom indicates the number of donors responding to the pooled HLA-DR-SPs. Increases of more than 60% above controls were considered positive. (D) Proliferation of HLA-DR-SP-specific bulk CD4 + T cells from HLA-DR15 + MS patients (n = 4) after co-culture with irradiated BLS-DR2a or BLS-DR2b cells and stimulation with the putative MS target antigens RASGRP2 (78–87) , MBP (13–32) , MBP (83–99) , MBP (111–129) , MBP (146–170) , PLP (139–154) , MOG (1–20) , and MOG (35–55) for 7 days (5 replicate wells). Groups with 2 or more positive wells are highlighted in red. (E) Proliferations of autoreactive TCC3A6, TCC5F6, or TCC14 after co-culture with irradiated BLS-DR2a or BLS-DR2b cells and stimulation with HLA-DR-SPs for 3 days. Responses to the cognate autoantigen are shown at the bottom. The purple dotted line indicates the mean + 3SD of the no-peptide control, and values above the purple dotted line were considered positive. (F) Th1/Th2/Th17-related cytokines in supernatants of TCC14 after co-culture with irradiated BLS-DR2b cells and stimulation with RASGRP2 (78–87) or HLA-DR-SPs for 3 days. (G) Proliferation of TCC14 after co-culture with irradiated BLS-DR2b cells and stimulation with HLA-DR-SPs for 3 days in the presence of anti-HLA-DR Ab. Data are expressed as mean ± SEM, and p values were determined by unpaired t test. See also Figure S5 and and .

Article Snippet: CD4 MicroBeads, human , Miltenyi Biotec , Cat# 130-045-101.

Techniques: Purification, Cell Culture, Irradiation, Co-Culture Assay, Control

Journal: Cell

Article Title: HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis

doi: 10.1016/j.cell.2020.09.054

Figure Lengend Snippet: HLA-DR-SP-Specific CD4 + T Cells in MS Patients Respond to Peptides of MBP, RASGRP2, and TSTA3, Related to and and (A) Workflow diagram for the isolation and expansion of the HLA-DR-SPs-specific bulk CD4 + T cells in HLA-DR15 + RRMS patients. (B) Proliferations of HLA-DR-SPs-specific bulk CD4 + T cells from HLA-DR15 + MS patients (n = 4) after co-culture with irradiated BLS-DR2a or BLS-DR2b cells as APCs and stimulation with peptide pools, including HLA-DR-SPs pool, myelin basic protein (MBP) peptide pools, myelin oligodendrocyte glycoprotein (MOG) peptide pools, myelin proteolipid protein (PLP) peptide pool, RASGRP2 peptide pools, and GDP-L-fucose synthase (TSTA3) peptide pools, for 7 days. Each stimulation was performed with 5 replicate wells. Proliferations were detected by 3 H-thymidine incorporation assay, and the proliferation strength is depicted as SI. Groups with 2 or more positive wells are highlighted in red. (C) The mRNA expression levels, expressed as FPKM (fragments per kilobase of exon model per million reads mapped), of RASGRP2 in TECs and blood B cells of HLA-DR15 + HDs. The purple dotted line indicates FPKM = 1. FPKM values above the purple dotted line were considered positive. (D) The mRNA expression levels of HLA-DRA, HLA-DRB1, and HLA-DRB5 in TECs and blood B cells of HLA-DR15 + HDs.

Article Snippet: CD4 MicroBeads, human , Miltenyi Biotec , Cat# 130-045-101.

Techniques: Isolation, Co-Culture Assay, Irradiation, Thymidine Incorporation Assay, Expressing

Journal: Cell

Article Title: HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis

doi: 10.1016/j.cell.2020.09.054

Figure Lengend Snippet: RASGRP2-Specific CD4 + T Cells Are Activated by Peptides from MS-Associated Foreign Agents (A and B) TCC14 was tested with predicted stimulatory decapeptides from the MS-associated pathogens EBV and Akkermansia (A) and control peptides from HCMV and Prevotella (B). Proliferation was detected on day 3. The x axis indicates the predicted stimulatory score of each peptide using the scoring matrix in Figure S6 A. (C and D) Irradiated BLS-DR2b cells were incubated with peptides for 12 h and then co-cultured with TCC14 for 24 h. Shown are upregulation of CD69 and CD25 and downregulation of TCR α/β on TCC14 (C) and Th1/Th2/Th17-related cytokines in supernatants (D). (E) Heatmap showing the top 1,000 high-variant genes that increased or decreased expression in TCC14 after co-culture with irradiated BLS-DR2b cells and stimulation with HLA-DR-SPs, peptides from EBV and Akkermansia , and RASGRP2 (78–87) for 24 h. (F) Expression levels of genes related to proliferation and Th2 cytokines (MKI67 [Ki-67], CCNE1 [Cyclin E1], IL-5, and IL-13), T cell activation (IL2RA [CD25], NFKBID [nuclear factor κB (NF-κB) inhibitor delta], CD40LG [CD40 ligand], and TNFRSF9 [4-1BB]) as well as cytotoxicity and MS pathogenesis (GZMB, FASLG, CSF2, and IL-3) in TCC14 after co-culture with irradiated BLS-DR2b cells and stimulation with HLA-DR-SPs, peptides from EBV and Akkermansia , and RASGRP2 (78–87) for 24 h. The mRNA expression levels are expressed as RPKM (reads per kilobase per million mapped reads). (G and H) 1159AG_TCCs were tested with predicted stimulatory decapeptides from EBV and Akkermansia . Proliferation was detected on day 3. Two positive peptides from Akkermansia are shown (G), as well as IFN-γ in supernatants (H). Data are expressed as mean or mean ± SEM. See also Figure S6 and .

Article Snippet: CD4 MicroBeads, human , Miltenyi Biotec , Cat# 130-045-101.

Techniques: Control, Irradiation, Incubation, Cell Culture, Variant Assay, Expressing, Co-Culture Assay, Activation Assay

Journal: Cell

Article Title: HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis

doi: 10.1016/j.cell.2020.09.054

Figure Lengend Snippet:

Article Snippet: CD4 MicroBeads, human , Miltenyi Biotec , Cat# 130-045-101.

Techniques: Purification, Recombinant, Produced, Staining, Lysis, Protease Inhibitor, Cell Isolation, Isolation, Enzyme-linked Immunosorbent Assay, Activation Assay, cDNA Synthesis, RNA Sequencing, Generated, Software