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: 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: Asian Journal of Pharmaceutical Sciences

Article Title: Lignin-assisted construction of sub-10 nm supramolecular self-assembly for photothermal immunotherapy and potentiating anti-PD-1 therapy against primary and distant breast tumors

doi: 10.1016/j.ajps.2022.07.002

Figure Lengend Snippet: In vivo immune responses assessment. (A) Flow cytometry assay of CD80 + or CD86 + gated on CD11c + in the TDLNs of mice. (B) Flow cytometry assay illustrated the percent of CD8a + T cells and CD4 + T cells gated on CD3 + T cells in splenocytes of mice. (C-D) Quantitative percentages of CD80 + or CD86 + expressions gated on CD11c + according to (A). (E) Quantitative percentages of CD8a + T cells and CD4 + T cells in splenocytes according to (B). (F-H) Quantitative analysis of the immune-associated cytokines in serum, as determined by ELISA (IL-2, TNF-α and IFN-γ). ( n = 3, mean ± SD, * P < 0.05, ** P < 0.01 and ## P < 0.0001).

Article Snippet: Fluorochrome-labeled anti-mouse monoclonal antibodies (CD11c-allophycocyanin (APC), CD80-fluorescein isothiocyanate (FITC), CD86-phycoerythrin (PE), CD3-FITC, CD8a-PE, and CD4-APC) were bought from Proteintech.

Techniques: In Vivo, Flow Cytometry, Enzyme-linked Immunosorbent Assay