Journal:

Article Title: Influence of Glycosylation on the Efficacy of an Env-Based Vaccine against Simian Immunodeficiency Virus SIVmac239 in a Macaque AIDS Model

doi: 10.1128/JVI.79.16.10386-10396.2005

Figure Lengend Snippet: Env-specific CD4+ T-cell and CD8+ T-cell responses in 12 macaques. A: Env-specific CD8+ T cells in PBMCs were measured by ELISPOT assay for IFN-γ in three groups. B: Env-specific CD4+ T cells in PBMCs were measured by ELISPOT assay for IFN-γ in three groups. ELISPOT results are colored as follows: Δ5G SU-specific T cells (red), wt-SU-specific T cells (green), and TM-specific T cells (yellow). Arrows with a dotted line, arrows with broken line, and arrows with a solid line indicate the time of the third DNA vaccination at 8 weeks p.p., the time of the vaccine boost at 21 weeks p.p., and the time of SIVmac239 challenge at 28 weeks p.p., respectively. C: Comparison of SU-specific CD8+ T cells and CD4+ T cells in PBMCs among the wt-Env vaccine group, the Δ5G Env vaccine group, and the vector control group at 26 weeks p.p. and 4, 7, and 12 days p.i. The numbers of SFC responding to SIVmac239 SU were used to compare the effects of the two vaccines. w, weeks; d, days.

Article Snippet: PBMCs were subjected to the depletion of CD4 + cells with magnet beads coated with anti-human CD4 Ab (Dynal ASA, Oslo, Norway) or subjected to the depletion of CD8 + cells with magnet beads coated with anti-human CD8 Ab (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Enzyme-linked Immunospot, Comparison, Plasmid Preparation, Vaccines

Journal:

Article Title: Influence of Glycosylation on the Efficacy of an Env-Based Vaccine against Simian Immunodeficiency Virus SIVmac239 in a Macaque AIDS Model

doi: 10.1128/JVI.79.16.10386-10396.2005

Figure Lengend Snippet: SIV-specific CD8+ T-cell and CD4+ T-cell responses in 12 animals. A: SIV viral-protein-specific CD8+ T cells in PBMCs were measured by ELISPOT assay for IFN-γ in three groups: vector controls, wt-Env vaccine group, and Δ5G Env vaccines. B: SIV viral-protein-specific CD4+ T cells in PBMCs were measured by ELISPOT assay for IFN-γ in three groups. ELISPOT results of individual SIV proteins are colored as follows: Gag (red), Nef (green), Tat/Rev (blue), Vif/Vpr/Vpx (yellow), and Pol (pink). C: Comparison of cumulated CD8+ T cells or CD4+ T cells specific to the viral proteins Gag, Pol, Nef, Tat/Rev, and Vif/Vpr/VpX between SIV infection-controlled and uncontrolled animals. w, weeks; d, days.

Article Snippet: PBMCs were subjected to the depletion of CD4 + cells with magnet beads coated with anti-human CD4 Ab (Dynal ASA, Oslo, Norway) or subjected to the depletion of CD8 + cells with magnet beads coated with anti-human CD8 Ab (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Enzyme-linked Immunospot, Plasmid Preparation, Vaccines, Comparison, Infection

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: Shown is the frequency of CFSE low CD4 T cells out of total CD4 T cells for all vaccinees, vaccinees per group and for each vaccinee. Peripheral blood mononuclear cells from day 60 were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with a pool of peptides spanning hepatitis B (HB) surface antigen (HBsAg) (peptide pool) and single peptides selected based on epitope mapping of the entire antigen (single peptide). After day 7 of in vitro expansion, cells were stained with antibodies to surface markers (CD3, CD4, and CD8) that enable gating on viable CD4 T cells. CFSE intensity was used to identify and sort CFSE low cells for T cell receptor (TCR) repertoire analysis of antigen-specific CD4 T cells.

Article Snippet: Cells were stained using the following fluorochrome-labeled monoclonal antibodies: CD3-PerCP (BW264/56), CD4-APC (REA623), and CD8-VioGreen (REA734) (purchased from Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Labeling, In Vitro, Staining

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: ( A ) Gating strategy started by a lymphocyte gate, followed by gating on viable CD3 + CD8 − T cells. Doublets were excluded using doublet discrimination (area against the height of forward scatter pulse) before gating on CD4 + T cells. Next, CD45RA, CXCR5, CD25, and CD127 were used to identify main subsets of CD4 T cells using Boolean gates as specified in the accompanying table. ( B ) Shown an example of gating for CD154 (CD40L) and CD137 (4-1BB) for cells left unstimulated (left) and cells stimulated with a master peptide pool (right) for an early-converter vaccinee at day 60.

Article Snippet: Cells were stained using the following fluorochrome-labeled monoclonal antibodies: CD3-PerCP (BW264/56), CD4-APC (REA623), and CD8-VioGreen (REA734) (purchased from Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques:

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet:

Article Snippet: Cells were stained using the following fluorochrome-labeled monoclonal antibodies: CD3-PerCP (BW264/56), CD4-APC (REA623), and CD8-VioGreen (REA734) (purchased from Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Recombinant, Sequencing, Software, Staining, Virus

Journal: Brain, Behavior, & Immunity - Health

Article Title: Acute exercise increases immune responses to SARS CoV-2 in a previously infected man

doi: 10.1016/j.bbih.2021.100343

Figure Lengend Snippet: The total number (cells/μL) of lymphocytes, CD3 + T-cells, CD4 + T-cells, CD8 + T-cells, ‘double-negative’ T-cells, NK-cells, B-Cells, and monocytes found in peripheral blood before (rest), during (60% and 80%), and 1 ​h after each exercise trial.

Article Snippet: EDTA whole blood samples collected from each timepoint were stained and analyzed by 7-color flow cytometry (MACSQuant 10; Miltenyi Biotec Inc., Germany) using the following directly conjugated antibodies: CD8-VioBlue, CD14-VioGreen, CD3-FITC, CD4-PE, CD20-PerCP, CD45-APC, CD56-APC-Vio770 (Miltenyi).

Techniques:

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: In vivo generation of CAR T cells in the presence of human myeloid cells

doi: 10.1016/j.omtm.2022.06.004

Figure Lengend Snippet: CAR T cell generation in huSGM3 mice (A–D) CD34+ cord blood humanized NSG-SGM3 (huSGM3) were injected intravenously (i.v.) with CD4-LV, CD8-LV, a mix of both (MIX), or PBS (Control). Mice received human IL-7 at 1 and 4 days before and 1 and 3 days after vector administration by i.v. or subcutaneous (s.c.) injection (A). Kinetic of CAR+ T cells and their CD19+ target cells in blood are shown as a CAR+ signal of CD8+ T cells (B), CD4+ T cells (C), and normalized B cells of CD3 cells (D) for each mouse. Dotted lines show the cutoff for determining reduction of B cells in mice. n = 4 (MIX), 8 (CD4-LV), 9 (CD8-LV), and 12 (Control) in two independent experiments. dpi, days post injection.

Article Snippet: The following anti-human antibodies were used for FACS staining: CD45 (2D1, BV510, BioLegend), CD3 (BW264/56, PerCP, Miltenyi Biotec; HIT3a, BV605, BD Biosciences), CD4 (VIT-4, FITC, Miltenyi Biotech; RPA-T4, PE-CF594, BD Biosciences, VIT-4, VioBlue, Miltenyi Biotech), CD8 (BW135/80, FITC, Miltenyi Biotech; RPA-T8, BV786, BD Biosciences; BW135/80, APC, Miltenyi Biotech), Myc for CAR detection (9B11, PE, Cell Signaling Technology; SH1-26e7.1.3, FITC, Miltenyi Biotech), CD19 (LT19, PE-Vio770, Miltenyi Biotech; HIB19, Alexa Fluor 700, Thermo Fisher), CD14 (REA599, APC, Miltenyi Biotech), CD206 (DCN228, VioBlue, Miltenyi Biotech), and CD209 (REA617, PE, Miltenyi Biotech).

Techniques: Injection, Control, Plasmid Preparation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: In vivo generation of CAR T cells in the presence of human myeloid cells

doi: 10.1016/j.omtm.2022.06.004

Figure Lengend Snippet: B cells and VCNs in spleen and bone marrow B cell frequencies and vector copy numbers (VCNs) of the CAR gene in the indicated organs. (A) B cell levels in the spleen and bone marrow are shown for the day of final analysis determined by FACS. (B) VCN per cell of the CAR transgene was measured by qPCR for the respective organ. The dotted lines represent the upper 95% confidence interval of the control group. Individual mice are plotted with mean and standard deviation of the group. n = 4 (MIX), 7–8 (CD4-LV), 9 (CD8-LV), and 12 (Control) in two independent experiments. Statistics were determined by non-parametric Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test and indicated significant p values compared with the Control.

Article Snippet: The following anti-human antibodies were used for FACS staining: CD45 (2D1, BV510, BioLegend), CD3 (BW264/56, PerCP, Miltenyi Biotec; HIT3a, BV605, BD Biosciences), CD4 (VIT-4, FITC, Miltenyi Biotech; RPA-T4, PE-CF594, BD Biosciences, VIT-4, VioBlue, Miltenyi Biotech), CD8 (BW135/80, FITC, Miltenyi Biotech; RPA-T8, BV786, BD Biosciences; BW135/80, APC, Miltenyi Biotech), Myc for CAR detection (9B11, PE, Cell Signaling Technology; SH1-26e7.1.3, FITC, Miltenyi Biotech), CD19 (LT19, PE-Vio770, Miltenyi Biotech; HIB19, Alexa Fluor 700, Thermo Fisher), CD14 (REA599, APC, Miltenyi Biotech), CD206 (DCN228, VioBlue, Miltenyi Biotech), and CD209 (REA617, PE, Miltenyi Biotech).

Techniques: Plasmid Preparation, Control, Standard Deviation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: In vivo generation of CAR T cells in the presence of human myeloid cells

doi: 10.1016/j.omtm.2022.06.004

Figure Lengend Snippet: Plasma cytokines in huSGM3 Plasma cytokines of huSGM3 mice on day 17 after vector administration were measured using a bead-based multi-analysis kit. Concentrations for the respective cytokines are shown for each mouse with mean and standard deviation within the group. n = 4 (MIX), 7 (CD4-LV), 8 (CD8-LV), and 11 (Control). Statistics were determined by non-parametric Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test and indicated significant p values.

Article Snippet: The following anti-human antibodies were used for FACS staining: CD45 (2D1, BV510, BioLegend), CD3 (BW264/56, PerCP, Miltenyi Biotec; HIT3a, BV605, BD Biosciences), CD4 (VIT-4, FITC, Miltenyi Biotech; RPA-T4, PE-CF594, BD Biosciences, VIT-4, VioBlue, Miltenyi Biotech), CD8 (BW135/80, FITC, Miltenyi Biotech; RPA-T8, BV786, BD Biosciences; BW135/80, APC, Miltenyi Biotech), Myc for CAR detection (9B11, PE, Cell Signaling Technology; SH1-26e7.1.3, FITC, Miltenyi Biotech), CD19 (LT19, PE-Vio770, Miltenyi Biotech; HIB19, Alexa Fluor 700, Thermo Fisher), CD14 (REA599, APC, Miltenyi Biotech), CD206 (DCN228, VioBlue, Miltenyi Biotech), and CD209 (REA617, PE, Miltenyi Biotech).

Techniques: Clinical Proteomics, Plasmid Preparation, Standard Deviation, Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: In vivo generation of CAR T cells in the presence of human myeloid cells

doi: 10.1016/j.omtm.2022.06.004

Figure Lengend Snippet: Reduced T cell transduction by macrophages is ameliorated by LV shielding (A) Normalized transduction of T cells co-cultivated with the indicated percentages of macrophages using CD4-LV (blue) or CD8-LV (green) produced in conventional packaging cells. (B) Comparison of conventional (blank bars) and shielded LVs (stripped bars) in transducing T cells in a 1:1 co-culture with (+) or without (O) macrophages. Mean with standard deviation from three to seven donors performed in four different experiments in technical triplicates. Statistics were determined by two-way ANOVA with Turkey’s (A) or Bonferroni’s (B) multiple comparisons test and indicated significant p values.

Article Snippet: The following anti-human antibodies were used for FACS staining: CD45 (2D1, BV510, BioLegend), CD3 (BW264/56, PerCP, Miltenyi Biotec; HIT3a, BV605, BD Biosciences), CD4 (VIT-4, FITC, Miltenyi Biotech; RPA-T4, PE-CF594, BD Biosciences, VIT-4, VioBlue, Miltenyi Biotech), CD8 (BW135/80, FITC, Miltenyi Biotech; RPA-T8, BV786, BD Biosciences; BW135/80, APC, Miltenyi Biotech), Myc for CAR detection (9B11, PE, Cell Signaling Technology; SH1-26e7.1.3, FITC, Miltenyi Biotech), CD19 (LT19, PE-Vio770, Miltenyi Biotech; HIB19, Alexa Fluor 700, Thermo Fisher), CD14 (REA599, APC, Miltenyi Biotech), CD206 (DCN228, VioBlue, Miltenyi Biotech), and CD209 (REA617, PE, Miltenyi Biotech).

Techniques: Transduction, Produced, Comparison, Co-Culture Assay, Standard Deviation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: In vivo generation of CAR T cells in the presence of human myeloid cells

doi: 10.1016/j.omtm.2022.06.004

Figure Lengend Snippet: In vivo CAR T cell generation using shielded LVs (A and B) huSGM3 mice were injected i.v. with the indicated vectors as a single or a double dose (2×). As control, PBS was injected into the mice. Kinetics of CAR+ T cells (A) in blood are shown as percentage CAR+ of respective T cell subtype and (B) normalized CD19+ cells of the CD3 population. Dotted lines show the cutoff for determining the reduction of B cells in mice. Mice determined as CAR– in blood are depicted with gray symbols and black connecting lines. n = 5 (CD4-LV), 6 (CD4-LV sh ), 5 (CD4-LV sh (2×)), 4 (CD8-LV), 5 (CD8-LV sh ), 5 (CD8-LV sh (2×)), and 4 (Control) in one experiment. dpi, days post injection.

Article Snippet: The following anti-human antibodies were used for FACS staining: CD45 (2D1, BV510, BioLegend), CD3 (BW264/56, PerCP, Miltenyi Biotec; HIT3a, BV605, BD Biosciences), CD4 (VIT-4, FITC, Miltenyi Biotech; RPA-T4, PE-CF594, BD Biosciences, VIT-4, VioBlue, Miltenyi Biotech), CD8 (BW135/80, FITC, Miltenyi Biotech; RPA-T8, BV786, BD Biosciences; BW135/80, APC, Miltenyi Biotech), Myc for CAR detection (9B11, PE, Cell Signaling Technology; SH1-26e7.1.3, FITC, Miltenyi Biotech), CD19 (LT19, PE-Vio770, Miltenyi Biotech; HIB19, Alexa Fluor 700, Thermo Fisher), CD14 (REA599, APC, Miltenyi Biotech), CD206 (DCN228, VioBlue, Miltenyi Biotech), and CD209 (REA617, PE, Miltenyi Biotech).

Techniques: In Vivo, Injection, Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: In vivo generation of CAR T cells in the presence of human myeloid cells

doi: 10.1016/j.omtm.2022.06.004

Figure Lengend Snippet: B cell depletion and VCNs in spleen and bone marrow (A) B cell levels in spleen and bone marrow at final analysis determined by FACS gated on CD19+ of human CD3 cells. (B) VCN of the CAR transgene measured by qPCR in enriched T cells from spleen. Dotted lines represent the upper standard deviation of the control group. X indicates datapoint below the axis. Individual mice are shown as data points with mean and standard deviation for n = 5 (CD4-LV), 6 (CD4-LV sh ), 5 (CD4-LV sh ([2×)), 4 (CD8-LV), 5 (CD8-LV sh ), 5 (CD8-LV sh (2×)), and 4 (Control) in one experiment. Statistics were determined by one-way ANOVA with Dunnett’s multiple comparisons test and indicated significant p values.

Article Snippet: The following anti-human antibodies were used for FACS staining: CD45 (2D1, BV510, BioLegend), CD3 (BW264/56, PerCP, Miltenyi Biotec; HIT3a, BV605, BD Biosciences), CD4 (VIT-4, FITC, Miltenyi Biotech; RPA-T4, PE-CF594, BD Biosciences, VIT-4, VioBlue, Miltenyi Biotech), CD8 (BW135/80, FITC, Miltenyi Biotech; RPA-T8, BV786, BD Biosciences; BW135/80, APC, Miltenyi Biotech), Myc for CAR detection (9B11, PE, Cell Signaling Technology; SH1-26e7.1.3, FITC, Miltenyi Biotech), CD19 (LT19, PE-Vio770, Miltenyi Biotech; HIB19, Alexa Fluor 700, Thermo Fisher), CD14 (REA599, APC, Miltenyi Biotech), CD206 (DCN228, VioBlue, Miltenyi Biotech), and CD209 (REA617, PE, Miltenyi Biotech).

Techniques: Standard Deviation, Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: In vivo generation of CAR T cells in the presence of human myeloid cells

doi: 10.1016/j.omtm.2022.06.004

Figure Lengend Snippet: Comparison of in vivo CAR T cell generation in huNSG and huSGM3 mice

Article Snippet: The following anti-human antibodies were used for FACS staining: CD45 (2D1, BV510, BioLegend), CD3 (BW264/56, PerCP, Miltenyi Biotec; HIT3a, BV605, BD Biosciences), CD4 (VIT-4, FITC, Miltenyi Biotech; RPA-T4, PE-CF594, BD Biosciences, VIT-4, VioBlue, Miltenyi Biotech), CD8 (BW135/80, FITC, Miltenyi Biotech; RPA-T8, BV786, BD Biosciences; BW135/80, APC, Miltenyi Biotech), Myc for CAR detection (9B11, PE, Cell Signaling Technology; SH1-26e7.1.3, FITC, Miltenyi Biotech), CD19 (LT19, PE-Vio770, Miltenyi Biotech; HIB19, Alexa Fluor 700, Thermo Fisher), CD14 (REA599, APC, Miltenyi Biotech), CD206 (DCN228, VioBlue, Miltenyi Biotech), and CD209 (REA617, PE, Miltenyi Biotech).

Techniques: Comparison, In Vivo, Plasmid Preparation

Journal: iScience

Article Title: TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion

doi: 10.1016/j.isci.2023.108401

Figure Lengend Snippet: Ascites blocks NK cell degranulation but not TRAIL-dependent HPMC apoptosis (A and B) TAL were pre-cultured in RPMI/5% AB media or 100% ascites pool (+/− α-CD3 Ab stimulation) prior to co-culture with HPMC. (A) Apoptosis of HPMC is shown as percentage of annexin V+ cells after gating on CD45 − cells (n = 7 patients). (B) Degranulating NK cells in response to HPMC were measured via flow cytometry and depicted as percentage of CD335+/CD107a+ cells (n = 6 patients). (C and D) TAL were pretreated with α-TRAIL blocking Ab prior to HPMC co-culture. An irrelevant mouse IgG was included as control. Experiments were conducted with TAL cultured in RPMI/5% AB media (n = 11 patients) (C) and 100% ascites pool (n = 5 patients) (D). The amount of Annexin V+ HPMC was determined as described previously. (E) Schematic representation of co-culture experiments applying T cell CM for NK cell activation. CM were collected from CD3 + , CD3+/CD4+, and CD3+/CD8+ T cell subsets cultured in media +/− α-CD3 Ab stimulation. Purified NK cells were then stimulated with T cell CM prior to HPMC co-cultures. (F) The amount of apoptotic HPMC induced by NK cells activated with CM of CD3 + T cells was compared with CM of CD3+/CD4+ and CD3+/CD8+ T cells (n = 4 matched pairs of different patients). (G) Analysis of TRAIL signaling was performed by applying an α-TRAIL blocking Ab to NK cells stimulated with CD3 + T cell CM (α-CD3 Ab activated) prior to the co-culture with HPMC (n = 5 patients). An irrelevant mouse IgG was included as control. The mean is shown by horizontal bars or boxes; vertical error bars represent the standard deviation in A–D, F, and G. ∗ FDR < 0.05; ∗∗ FDR < 0.01; ∗∗∗ FDR < 0.001; determined by paired t test and Benjamini-Hochberg adjustment (ns, not significant).

Article Snippet: To further divide the CD3 + T cell fraction into CD3+/CD4+ T helper cells and CD3+/CD8+ cytotoxic T-cells, the preselected CD14 − lymphocytes were stained with APC-labelled anti-human CD8 antibody (Miltenyi Biotec), incubated with anti-APC microbeads and isolated via MACS.

Techniques: Cell Culture, Co-Culture Assay, Flow Cytometry, Blocking Assay, Control, Activation Assay, Purification, Standard Deviation

Journal: iScience

Article Title: TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion

doi: 10.1016/j.isci.2023.108401

Figure Lengend Snippet:

Article Snippet: To further divide the CD3 + T cell fraction into CD3+/CD4+ T helper cells and CD3+/CD8+ cytotoxic T-cells, the preselected CD14 − lymphocytes were stained with APC-labelled anti-human CD8 antibody (Miltenyi Biotec), incubated with anti-APC microbeads and isolated via MACS.

Techniques: Control, Recombinant, Polymer, Software