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: Cancer cell

Article Title: Lymphoma accelerates T cell and tissue aging

doi: 10.1016/j.ccell.2025.07.023

Figure Lengend Snippet: (A) Experimental design analyzing the effects of B cell lymphoma on young and aged immune systems. (B) Flow cytometry gating strategy for CD4 + T cells, CD8 + T cells, NK1.1 + NK cells, B220 lo FSC-A hi lymphoma cells, and B220 hi FSC-A lo normal B cells. (C) Total splenocyte number ( n = 7). (D) Number of B220 lo FSC-A hi lymphoma cells ( n = 7). (E–I) Number of splenic cells ( n = 3–7): (E) Ter119 + cells; (F) CD8 + T cells; (G) CD4 + T cells; (H) normal B cells; and (I) NK cells. YC = young control; YL = young with lymphoma, AC = aged control, AL = aged with lymphoma. Data are mean ± SEM and are analyzed by a Student’s t test except for a one-way ANOVA and Tukey’s test (C and E). Data are compiled from at least two independent experiments and each dot represents an individual mouse. ** p < 0.01, *** p < 0.001, and **** p < 0.0001, n.s. = not significant.

Article Snippet: CD8 – FITC, human , Tonbo , Cat # 35-0089; RRID:AB_2621675.

Techniques: Flow Cytometry, Control

Journal: Cancer cell

Article Title: Lymphoma accelerates T cell and tissue aging

doi: 10.1016/j.ccell.2025.07.023

Figure Lengend Snippet: (A) Intracellular iron measured by BioTracker Fe 2+ in CD4 + and CD8 + T cells ( n = 6–14). (B) Intracellular iron measured by BioTracker Fe 2+ in CD44/CD62L CD4 + T cells memory populations ( n = 6–11). (C) Intracellular iron measured by BioTracker Fe 2+ in CD4 + T cells from OT-II mice ( n = 6). (D) Intracellular iron measured by BioTracker Fe 2+ in human CD4 + and CD8 + T cells from young and aged healthy donors or from R/R LBCL patients ( n = 5–6). (E) Percent DAPI − viable cells of YC or AC CD4 + and CD8 + T cells unstimulated or activated overnight ±10 mM ferric ammonium citrate (FAC) ( n = 3). (F) Aifm2 and Gpx4 gene expression in young and aged T cells activated overnight ( n = 3–4). (G) Percent DAPI − Annexin V − viable cells from human CD4 + (left) and CD8 + (right) T cells unstimulated or activated for 5 days ±10 mM FAC ( n = 3). (H) Percent DAPI − viable cells of YC or YL CD4 + and CD8 + T cells unstimulated or activated overnight ±10 mM FAC ( n = 3). (I) Aifm2 and Gpx4 gene expression in young T cells from control and lymphoma-bearing mice activated overnight ( n = 4). (J) ATAC-seq track around the Aifm2 locus and the accessibility index for the highlighted regions. (K) Experimental design for the A20 lymphoma BALB/cJ model. (L) Intracellular iron measured by BioTracker Fe 2+ in CD4 + and CD8 + T cells from control and A20 lymphoma-bearing BALB/cJ mice ( n = 4–8). (M) Total splenocyte number in control and A20 lymphoma-bearing mice ( n = 4–8). (N) Total splenic CD4 + and CD8 + T cells from control and A20 lymphoma-bearing mice ( n = 4–8). YC, YL, AC, AL are defined in . Data are mean ± SEM (A–D, L–N) or are mean ± SD (E–I). Data (A–D, K–N) are compiled from at least two independent experiments (2–6) and each solid dot represents a biological replicate; or data (E–I) were repeated at least twice with similar result and each hollow dot represents a technical replicate. Data were analyzed by a Student’s t test (A, C, D, F, I, and L–N). For (B, E, G, and H), data were analyzed by a two-way ANOVA with a Tukey’s test or Sidak’s test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001; n.s. = not significant. See also .

Article Snippet: CD8 – FITC, human , Tonbo , Cat # 35-0089; RRID:AB_2621675.

Techniques: Gene Expression, Control

Journal: Cancer cell

Article Title: Lymphoma accelerates T cell and tissue aging

doi: 10.1016/j.ccell.2025.07.023

Figure Lengend Snippet: (A and B) Cdkn2a and Tnfa expression in aorta and kidney tissues ( n = 3–5). (C and D) Cdkn2a expression in the aorta and kidney tissues of young control and PPMBC lymphoma-bearing Rag2 −/− mice ( n = 3–5). (E) Gene set enrichment analysis of the SenMayo pathways in young CD4 + T cells derived from lymphoma-bearing mice early vs. late following Eμ- Myc lymphoma transplant ( GSE183693 ). (F) Relative gene expression of SenMayo pathway in CD4 + T cells from bulk RNA-seq data ( GSE183693 ). (G) Cdkn2a and Grem2 gene expression in CD4 + T cells from RNA-seq data ( GSE183693 ). (H) CDKN2A gene expression in human T cells from healthy PBMCs (young, aged) or from apheresis products of young or aged R/R LBCL patients ( n = 4–5). (I–M) ATAC-seq tracks of the (I) Cdkn2a , (J) Serpine1 , (K) Timp2 , (L) Ifng , and (M) Tnfaip2 loci and the accessibility index for the highlighted regions. (N) TNFα production in mouse CD4 + and CD8 + T cells ( n = 3–7). (O) TNFα production in human CD4 + T cells and CD8 + T cells ( n = 5–6). (P and Q) NIAD4 staining in (P) CD4 + and CD8 + T cells ( n = 5–8) or (Q) OT-II CD4 + T cells ( n = 6). (R and S) ER Tracker in (R) CD4 + and CD8 + T cells ( n = 5–8) or (S) OT-II CD4 + T cells ( n = 6). YC, YL, AC, AL are defined in . YL-E = young with lymphoma early (day 7); YL-L = young with lymphoma late (day 14). Data in (A–D), (G), (H), and (N–S) are mean ± SEM and each solid dot represents a biological replicate. Data in (A–D), (N), and (P–S) are compiled from at least 2 independent experiments. For (A–D), (G), (H), (N–S), statistical significance was determined by Student’s t test. * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001; n.s. = not significant. See also .

Article Snippet: CD8 – FITC, human , Tonbo , Cat # 35-0089; RRID:AB_2621675.

Techniques: Expressing, Control, Derivative Assay, Gene Expression, RNA Sequencing, Staining

Journal: Cancer cell

Article Title: Lymphoma accelerates T cell and tissue aging

doi: 10.1016/j.ccell.2025.07.023

Figure Lengend Snippet: (A) Schematic for generating and using Eμ- Myc ; Cd19-Cre ;iDTR lymphomas that can be depleted by DT treatment of lymphoma-bearing mice. (B) Flow cytometry gating strategy for B220 lo FSC-A hi lymphoma cells from untreated or diphtheria toxin (DT) (300 ng) treated mice. (C) Number of B220 lo FSC-A hi lymphoma cells at endpoint ( n = 3–4/group). (D) Total splenocyte number ( n = 3–4). (E) Number of splenic CD4 + and CD8 + T cells from YC, YL, and YL + DT mice ( n = 3–4). (F) BioTracker Fe 2+ in the indicated CD4 + and CD8 + T cells ( n = 3–4). (G) ER Tracker in CD4 + and CD8 + T cells ( n = 3–4). (H) NIAD-4 staining in CD4 + and CD8 + T cells ( n = 3–4). (I) TNFα production in CD4 + and CD8 + T cells ( n = 3–4). (J) IFNγ production in CD4 + T cells (n = 3–4). (K) Naive, effector, central memory, and CD62L + CD44 lo CD4 + T cell populations ( n = 3–4). (L and M) Cdkn2a and Tnf expression in aortas and kidneys relative to Gapdh ( n = 5–7). YC = young control; YL = young with Eμ- Myc ;DTR; YL + DT = young with Eμ- Myc ;DTR and treated with 300 ng DT prior to endpoint. Data are mean ± SEM and each point represents a biological replicate. Data are analyzed by a Student’s t test (C), a one-way ANOVA with Dunnett’s multiple comparison test (D–J, L, M), or a two-way ANOVA with Sidak’s multiple comparisons test (K). Data (L and M) are compiled from two experiments or represented of two independent experiment (C–K); each dot represents an individual mouse. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, n.s. = not significant.

Article Snippet: CD8 – FITC, human , Tonbo , Cat # 35-0089; RRID:AB_2621675.

Techniques: Flow Cytometry, Staining, Expressing, Control, Comparison