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: Clinical and Translational Medicine

Article Title: PCIF1 drives oesophageal squamous cell carcinoma progression via m6Am‐mediated suppression of MTF2 translation

doi: 10.1002/ctm2.70286

Figure Lengend Snippet: PCIF1 knockout enhances the efficacy of anti‐PD1 therapy in oesophageal squamous cell carcinoma (OSCC). (A) Schematic of the experimental design involving the generation of PCIF1 knockout in OSCC mouse models, followed by treatment with anti‐PD1 antibody or IgG control. Mice were administered 4NQO in drinking water to induce tumour formation. Anti‐PD1 or IgG treatment was administered starting from Week 22, and mice were sacrificed at Week 26 for analysis. (B) Representative images of oesophageal tumours from different treatment groups: IgG control, Anti‐PD1 treated and combined anti‐PD1 + PCIF1 knockout. Scale bar, 2 mm. (C) Quantification of lesion area, showing a reduction in tumour size in the PCIF1 knockout group and in the combined anti‐PD1 + PCIF1 knockout group compared to controls. ** p < .01, *** p < .001 by one‐way analysis of variance (ANOVA) with Tukey's multiple comparison test. (D) Haematoxylin and eosin (H&E) staining of oesophageal tissue sections from each treatment group. Scale bar, 200 µm. (E) Quantification of the number of lesions, indicating a significant reduction in the combined treatment group. * p < .05 by one‐way ANOVA with Tukey's multiple comparison test. (F) Tumour invasion grade analysis, showing decreased aggressiveness in the anti‐PD1 + PCIF1 knockout group. * p < .05 by Pearson chi‐square test. (G and H) Immunohistochemistry (IHC) staining (G) and quantification (H) of PCIF1‐positive cells across the treatment groups, showing a marked reduction in PCIF1 expression in the knockout group. Scale bar, 100 µm. *** p < .001 by one‐way ANOVA with Tukey's multiple comparison test. (I and J) IHC staining (I) and quantification (J) of MTF2‐positive cells, with increased MTF2 expression in the Anti‐PD1 + PCIF1 knockout group. Scale bar, 100 µm. *** p < .001 by one‐way ANOVA with Tukey's multiple comparison test. (K and L) IHC staining (K) for Ki67 with quantification (L) showing decreased Ki67‐positive cells in the anti‐PD1 and PCIF1 knockout groups, particularly in the combined treatment group, indicating reduced tumour proliferation. Scale bar, 100 µm. *** p < .001 by one‐way ANOVA with Tukey's multiple comparison test. (M–P) Flow cytometry analysis showing no significant changes in CD8 + T cell function across the treatment groups, including FPR1+ and GZMB+ CD8 + T cells, suggesting that PCIF1 knockout and anti‐PD1 treatment operate via distinct mechanisms. p > .05, *** p < .001 by Student's t ‐test.

Article Snippet: The resulting single‐cell suspensions were stained with anti‐mouse CD3 APC (20–0032; Tonbo; 1:200), anti‐mouse CD8a FITC (35–0081;Tonbo; 1:200), anti‐Fpr1 (NLS1878; Novus) and anti‐GZMB PE (12‐8898‐82;Invitrogen; 1:200).

Techniques: Knock-Out, Control, Comparison, Staining, Immunohistochemistry, Expressing, Flow Cytometry, Cell Function Assay

Journal: iScience

Article Title: Akkermansia muciniphila ameliorates fatty liver through microbiota-derived α-ketoisovaleric acid metabolism and hepatic PI3K/Akt signaling

doi: 10.1016/j.isci.2025.112458

Figure Lengend Snippet: Akk improves gut immune responses in obese mice (A) PHATE of T cells clustering in mice intestinal lymph nodes. (B–E) Percentages of CD3 + CD4 + Tregs, CD3 + CD8 + Tregs, CD4 + CD25 + Tregs, and CD4 + CD25 + Foxp3 + Tregs. Data are expressed as mean ± SD ( n = 6), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, compared with the Mod group, and p value was calculated using Dunnet’s post hoc test in one-way ANOVA.

Article Snippet: Anti-Mouse CD8 + FITC , BioGems , Cat# 10112-50.

Techniques:

Journal: iScience

Article Title: Akkermansia muciniphila ameliorates fatty liver through microbiota-derived α-ketoisovaleric acid metabolism and hepatic PI3K/Akt signaling

doi: 10.1016/j.isci.2025.112458

Figure Lengend Snippet: Gut fungi is the key in Akk improving gut immune responses in organoids (A) Co-culture of colonic organoids, fecal microbiota and Akk. (B) Fluorescent images of colonic organoids with DAPI-labeled cells, and number of organoid crypts. Scale bars: 100 μm. (C) Relative abundance of fungal in co-culture system. (D) Percentages of CD3 + CD4 + , CD3 + CD8 + , CD4 + CD25 + , and CD4 + CD25 + Foxp3 + Tregs in organoids. (E) Relative content of metabolites in co-culture system. (F) Co-culture of colonic organoids, fluconazole, fecal microbiota and Akk. (G) Fluorescent images of colonic organoids with DAPI-labeled cells, and number of organoid crypts. Scale bars: 50 μm. (H) Percentages of CD3 + CD4 + , CD3 + CD8 + , CD4 + CD25 + , and CD4 + CD25 + Foxp3 + Tregs in organoids. (I and J) Relative content of metabolites (α-ketoisovaleric acid and glycylleucine) in co-culture system. Figures 7 A–7E and Figures 7 F–7J represent experiments from different batches, respectively. Data are expressed as mean ± SD (n = 6–8), ns p >0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, compared with the Mod group, and p value was calculated using Dunnet’s post hoc test in one-way ANOVA.

Article Snippet: Anti-Mouse CD8 + FITC , BioGems , Cat# 10112-50.

Techniques: Co-Culture Assay, Labeling