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