ngfr o4  (Miltenyi Biotec)

Journal: Developmental Dynamics

Article Title: Combinatorial expression of glial transcription factors induces Schwann cell‐specific gene expression in mouse embryonic fibroblasts

doi: 10.1002/dvdy.70054

Figure Lengend Snippet: Analysis of Schwann cell mRNA marker expression in NGFR + O4 + Schwann‐like cells isolated from NGFR‐negative MEFs transfected with negative, Sox10‐, T1‐ or T2‐expression vectors. (A) Schematic of the lineage conversion strategy, with a negative FACS sort to remove NGFR + Schwann cell lineage cells contaminating MEFs prior to transfection. Cells were analyzed by qPCR at 21‐days post‐transfection. (B) FACS plot showing the NGFR‐negative cell population sorted out (red asterisk). (C) Schematic showing the isolation of nerve‐derived Schwann cells, used as a positive control for qPCR. (D) Relative Sox10, Sox2, Pax3 and Jun transcript levels in negative control and Sox10‐, T1‐ and T2‐transfected NGFR‐negative MEFs, assessed by qPCR at 21‐days post‐transfection. N = 3. Graphs show means ± s.e.m. Statistical comparisons were made using an ANOVA with a post‐hoc Tukey's test. (E) Relative Ngfr, Gap43, Tfap2a, Cdh19, Egr1, Egr2, S100b, Mpz, Olig1 , and Col1a1 transcript levels in negative control and Sox10‐, T1‐ and T2‐transfected NGFR‐negative MEFs, assessed by qPCR at 21‐days post‐transfection. N = 3. Graphs show means ± s.e.m. Statistical comparisons were made using an ANOVA with a post‐hoc Tukey's test. (F) FACS plot, showing the fluorescence intensity of O4 and NGFR labeling of MEFs transfected with negative, Sox10, T1 and T2 expression vectors after 21 days. NGFR + O4 + cells were sorted out, including O4 + cells with or without NGFR expression.

Article Snippet: To isolate NGFR + O4 + reprogrammed cells, dissociated cells were incubated with CD271‐VioBright FITC (Miltenyi Biotech; 130‐110‐115) and O4‐APC (Miltenyi Biotech, 130‐117‐357) antibodies.

Techniques: Marker, Expressing, Isolation, Transfection, Derivative Assay, Positive Control, Negative Control, Fluorescence, Labeling

Journal: Developmental Dynamics

Article Title: Combinatorial expression of glial transcription factors induces Schwann cell‐specific gene expression in mouse embryonic fibroblasts

doi: 10.1002/dvdy.70054

Figure Lengend Snippet: Schwann cell protein marker expression in NGFR‐negative MEFs transfected with negative, Sox10‐, T1‐ or T2‐expression vectors. (A) Schematic of the lineage conversion strategy, adding a negative FACS sort to remove NGFR + Schwann cell lineage cells prior to transfection. Cells were analyzed by NGFR and GAP43 immunostaining and O4 + flow cytometry at 21‐days post‐transfection. (B) Immunostaining of GAP43 and NGFR on NGFR‐negative MEFs transfected with Sox10 , T1, and T2 after 21‐days in vitro (DIV). DAPI counterstain in blue. (Scale bar = 100 μm). (C) Flow cytometry to assess O4 expression in unstained MEFs (to set gate) and NGFR‐negative MEFs transfected with Sox10, T1, or T2 after 21‐days. The percentages of cells in the O4 + gate are shown.

Article Snippet: To isolate NGFR + O4 + reprogrammed cells, dissociated cells were incubated with CD271‐VioBright FITC (Miltenyi Biotech; 130‐110‐115) and O4‐APC (Miltenyi Biotech, 130‐117‐357) antibodies.

Techniques: Marker, Expressing, Transfection, Immunostaining, Flow Cytometry, In Vitro

Journal: Developmental Dynamics

Article Title: Combinatorial expression of glial transcription factors induces Schwann cell‐specific gene expression in mouse embryonic fibroblasts

doi: 10.1002/dvdy.70054

Figure Lengend Snippet: Reprogrammed Schwann cells express some endogenous Schwann cell repair genes and show some myelinating capacity. (A) Expression of Col1a, Thy1, Olig1, S100b, Cdh19, Ngfr, Mbp, Mpz, Gap43, Pax3, Tfap2a, Egr1 and Egr2 at 3, 5 and 7‐days post‐PNI ⁶³ (from GSE177037 ). Arrows show genes that were induced in reprogrammed Schwann cells, either with all of T1, T2 and Sox10 (green arrows), or induced by higher levels by T1 (red arrows) or Sox10 (blue arrow). (B) Dotplot showing the expression levels of Col1a, Thy1, Olig1, S100b, Cdh19, Ngfr, Mbp, Mpz, Gap43, Pax3, Tfap2a, Egr1 and Egr2 in Schwann cell subtypes in naïve Schwann cells and at 3, 5 and 7‐days post‐injury ⁶³ (from GSE177037 ). Arrows show genes that were induced in reprogrammed Schwann cells, either with all of T1, T2 and Sox10 (green arrows), or induced by higher levels by T1 (red arrows) or Sox10 (blue arrow). (C, D) Schematic of DRG‐Schwann cell co‐culture assay. P2 wild‐type DRGs were co‐cultured with tdTomato‐labeled Schwann cells collected from P21 Sox10‐cre;Rosa‐tdTomato nerves (C). Immunostaining of DRG‐ Schwann cell co‐culture after 15 days with tdTomato (labeling Schwann cells in red) and TUJ1 (labeling DRG neurite outgrowth in green) and DAPI counterstain in blue (D). (Scale bars: 100 μm [top left images], the rest were digitally magnified at 75 and 50 μm).

Article Snippet: To isolate NGFR + O4 + reprogrammed cells, dissociated cells were incubated with CD271‐VioBright FITC (Miltenyi Biotech; 130‐110‐115) and O4‐APC (Miltenyi Biotech, 130‐117‐357) antibodies.

Techniques: Expressing, Co-culture Assay, Cell Culture, Labeling, Immunostaining, Co-Culture Assay

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Design considerations for single and dual-receptor engineered T cells (A) Schematic representation of a DAP12-associated synthetic antigen receptor (DAP12-SAR) composed of an antigen binding domain fused to the hinge, transmembrane (TM) and intracellular (ICD) domains of a DAP12-associated activating receptor (created using BioRender). (B) Schematic diagram of cDNA encoding DAP12 and the SAR separated by a Thoseasigna virus 2A (T2A) sequence for co-expression. (C) SAR surface expression was determined by binding of a myc-tag specific mAb or HER2-Fc to T cells engineered with the IL13Rα2-KIR and HER2-KIR, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + . Presented is SAR expression on the CD4 + NGFR + population (unfilled black = non-transduced; gray = SAR). (D) Representative cytotoxicity of IL13Rα2-KIR, HER2-KIR and non-specific SAR T cell products after 96-h co-culture with U-251 tumor cells in an Incucyte assay (E:T = 8:1). The experiment was performed in technical triplicates and error bars are standard error mean (SEM). (E) Schematic representation of dual-SAR constructs. (F) SAR surface expression on T cells engineered with single and dual SAR constructs, using anti-Myc tag mAb and HER2-Fc to detect binding of the IL13Rα2-KIR and HER2-KIR, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + .

Article Snippet: All proliferation assay samples were incubated for 3 days at 37°C and stained with Live/Dead Fixable Near-IR stain (Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (eBioscience) and VioBright FITC-conjugated mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Binding Assay, Virus, Sequencing, Expressing, Co-Culture Assay, Construct

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Dual targeting of IL13Rα2 and HER2 can be achieved by expression of different synthetic DAP12-associated receptors (A) Schematic representation of various HER2-SAR constructs evaluated. (B) Receptor surface expression and transduction efficiency of various HER2-SAR constructs, determined by binding to HER2-Fc and tNGFR expression, respectively. (C) Cytotoxicity of HER2-SAR engineered T cells after 120 h co-culture with HCT-116 and U-251 tumor cells in an Incucyte assay. (D) Proliferation of respective HER2-SAR constructs after 72 h co-culture with HCT-116 or U-251 tumor cells at a 1:1 ratio. Absolute cell count was determined by flow cytometry using 123count eBeads. (E) Schematic diagram of cDNA encoding single and dual SAR constructs. (F) SAR surface expression of single and dual IL13Rα2/HER2 SAR T cells determined by binding of a myc-tag specific mAb or HER2-Fc to detect the IL13Rα2-KIR and HER2-TREM1 SARs, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + . Presented is SAR expression on the CD4 + NGFR + population. (G) Mean fluorescence intensity (MFI) of respective SARs on single and dual-receptor engineered T cells. Data are from 4 experiments with 4 PBMC donors (each donor is represented by a unique symbol). (H) Cytotoxicity of single, dual, and non-transduced T cell products after 120-h co-culture with U-251 tumor cells in an Incucyte assay. All conditions were normalized to growth of tumor cells alone. Data are from 4 individual experiments with 3 PBMC donors. Error bars represent SEM of technical replicates. (I) Cytotoxicity of single, dual, and non-transduced T cell products after 120-h co-culture with U-251 IL13Rα2 KO and U-251 HER2 KO cells in an IncuCyte assay. Data are representative of 3 experiments with 3 PBMC donors. Error bars represent SEM. Statistical analysis for (G) and (H) were performed using a paired t test and two-way ANOVA with correction for multiple comparison (Tukey test), respectively (∗ p = ≤ 0.05, ∗∗ p = ≤ 0.01, ∗∗∗ p = ≤ 0.001, ∗∗∗∗ p = ≤ 0.0001).

Article Snippet: All proliferation assay samples were incubated for 3 days at 37°C and stained with Live/Dead Fixable Near-IR stain (Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (eBioscience) and VioBright FITC-conjugated mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Expressing, Construct, Transduction, Binding Assay, Co-Culture Assay, Cell Counting, Flow Cytometry, Fluorescence, Comparison

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Combinatorial targeting of CD133 and HER2 can be achieved using the dual-SAR approach (A) Schematic diagram of cDNA encoding CD133-SAR constructs. (B) SAR surface expression on primary human T cells as determined by binding of a myc-tag specific mAb or HER2-Fc to detect the IL13Rα2-KIR and HER2-TREM1 SARs, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + . (C) T cells were incubated with firefly luciferase-expressing HCT-116 tumor cells for 20 h at indicated effector:target ratios. Luminescence was read with an open filter upon addition of 0.15 mg/mL D-luciferin substrate and converted to % cytotoxicity. Error bars display standard deviation for technical replicates. (D) T cells were CTV-labeled and incubated for 72-h with HCT-116 tumor cells at a 1:1 ratio. T cell proliferation was measured by flow cytometry with live > CD3 + > CD4 + > NGFR+ cells presented. (E) Schematic diagram of cDNA encoding single and dual CD133/HER2 SAR constructs. (F) SAR surface expression of single and dual CD133/HER2 SAR T cells determined by binding of a FLAG tag specific mAb or HER2-Fc to detect the CD133-NKp44 and HER2-TREM1 SARs, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + . Presented is SAR expression on the CD4 + NGFR + population. (G) Mean fluorescence intensity (MFI) of respective SARs on single and dual-receptor engineered T cells. Data are from 4 experiments with 4 PBMC donors (each donor is represented by a unique symbol). (H) Cytotoxicity of single, dual, and non-transduced T cell products after 72-h co-culture with HCT-116 tumor cells in an Incucyte assay. All conditions in were normalized to growth of tumor cells alone. Data are from 4 individual experiments with 3 PBMC donors. Error bars represent SEM. (I) Cytotoxicity of single, dual and non-transduced T cell products after 120-h co-culture with HCT-116 CD133 KO and HCT-116 HER2 KO cells in an IncuCyte assay. Data are representative of 3 experiments with 3 PBMC donors. Error bars represent SEM of technical replicates. Statistical analysis for (G) and (H) were performed using a paired t test and two-way ANOVA with correction for multiple comparison (Tukey test), respectively (∗ p = ≤ 0.05, ∗∗ p = ≤ 0.01, ∗∗∗ p = ≤ 0.001, ∗∗∗∗ p = ≤ 0.0001).

Article Snippet: All proliferation assay samples were incubated for 3 days at 37°C and stained with Live/Dead Fixable Near-IR stain (Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (eBioscience) and VioBright FITC-conjugated mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Construct, Expressing, Binding Assay, Incubation, Luciferase, Standard Deviation, Labeling, Flow Cytometry, FLAG-tag, Fluorescence, Co-Culture Assay, Comparison

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Expression of multiple receptors attenuates the production of inflammatory cytokines but not proliferation (A and D) Schematic representation of stimulation conditions for single- vs. dual-SAR engagement against (A) U-251 tumor cells or (D) HCT-116 tumor cells. (B and E) Intracellular cytokine production by (B) U-251 or (E) HCT-116 stimulated engineered T cells was measured by flow cytometry. Data are presented as percent NGFR+ CD4 and CD8 T cells producing respective cytokines. Data are from four independent experiments with 3 PBMC donors. Each donor is represented by a unique symbol. For the gating strategy see Figure S6 A. (C and F) Engineered T cells were labeled with CellTrace Violet (CTV) and stimulated with (C) U-251 or (F) HCT-116 tumor cells at a 1:1 ratio for 72 h. T cell proliferation and absolute cell count using 123count eBeads were measured by flow cytometry. Data are from three independent experiments with 2 T cell donors. Each donor is represented by a unique symbol. For the gating strategy see Figure S6 . Statistical analysis for (B), (C), (E), and (F) were performed using two-way ANOVA with correction for multiple comparison (Tukey test) (∗ p = ≤ 0.05, ∗∗ p = ≤ 0.01, ∗∗∗ p = ≤ 0.001, ∗∗∗∗ p = ≤ 0.0001, ns = not significant.

Article Snippet: All proliferation assay samples were incubated for 3 days at 37°C and stained with Live/Dead Fixable Near-IR stain (Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (eBioscience) and VioBright FITC-conjugated mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Expressing, Flow Cytometry, Labeling, Cell Counting, Comparison

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Expression of multiple receptors attenuates the early signal strength of individual receptors (A and B) Intracellular phospho-specific staining of ERK. Cells were stimulated for 30 min with respective WT or KO tumor lines, fixed, methanol-permeabilized and stained for phosphorylated ERK 1/2 (pT202/pY204). (A) Representative plots from 1 of 4 independent experiments and (B) %pERK+ of single and dual-SAR T cells following stimulation with a single target antigen. Data are from 4 independent experiments and 3 T cell donors. (C) Cells were stimulated with HCT-116 CD133 KO or U251 IL13Rα2 KO tumor cells for 1–4 h. The cells were surface stained for viability, CD4, CD8, NGFR and CD69, fixed/permeabilized, and then stained intracellularly/intranuclearly for Nur77. Cells were gated as follows: lymphocytes > single cells > live > CD4/CD8 > NGFR + > Nur77/CD69. Presented is Nur77 and CD69 expression on the CD8 + NGFR + population. Data are generated with T cells from one donor. For the gating strategy, see Figure S6 C.

Article Snippet: All proliferation assay samples were incubated for 3 days at 37°C and stained with Live/Dead Fixable Near-IR stain (Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (eBioscience) and VioBright FITC-conjugated mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Expressing, Staining, Generated

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Design considerations for single and dual-receptor engineered T cells (A) Schematic representation of a DAP12-associated synthetic antigen receptor (DAP12-SAR) composed of an antigen binding domain fused to the hinge, transmembrane (TM) and intracellular (ICD) domains of a DAP12-associated activating receptor (created using BioRender). (B) Schematic diagram of cDNA encoding DAP12 and the SAR separated by a Thoseasigna virus 2A (T2A) sequence for co-expression. (C) SAR surface expression was determined by binding of a myc-tag specific mAb or HER2-Fc to T cells engineered with the IL13Rα2-KIR and HER2-KIR, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + . Presented is SAR expression on the CD4 + NGFR + population (unfilled black = non-transduced; gray = SAR). (D) Representative cytotoxicity of IL13Rα2-KIR, HER2-KIR and non-specific SAR T cell products after 96-h co-culture with U-251 tumor cells in an Incucyte assay (E:T = 8:1). The experiment was performed in technical triplicates and error bars are standard error mean (SEM). (E) Schematic representation of dual-SAR constructs. (F) SAR surface expression on T cells engineered with single and dual SAR constructs, using anti-Myc tag mAb and HER2-Fc to detect binding of the IL13Rα2-KIR and HER2-KIR, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + .

Article Snippet: Other phenotypic markers were detected with mouse anti-human CD4 AlexaFluor700 (Invitrogen), mouse anti-human CD8a PerCP-Cyanine5.5 (Invitrogen), mouse anti-human CD8 BV786 (BD Biosciences) and mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Binding Assay, Virus, Sequencing, Expressing, Co-Culture Assay, Construct

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Dual targeting of IL13Rα2 and HER2 can be achieved by expression of different synthetic DAP12-associated receptors (A) Schematic representation of various HER2-SAR constructs evaluated. (B) Receptor surface expression and transduction efficiency of various HER2-SAR constructs, determined by binding to HER2-Fc and tNGFR expression, respectively. (C) Cytotoxicity of HER2-SAR engineered T cells after 120 h co-culture with HCT-116 and U-251 tumor cells in an Incucyte assay. (D) Proliferation of respective HER2-SAR constructs after 72 h co-culture with HCT-116 or U-251 tumor cells at a 1:1 ratio. Absolute cell count was determined by flow cytometry using 123count eBeads. (E) Schematic diagram of cDNA encoding single and dual SAR constructs. (F) SAR surface expression of single and dual IL13Rα2/HER2 SAR T cells determined by binding of a myc-tag specific mAb or HER2-Fc to detect the IL13Rα2-KIR and HER2-TREM1 SARs, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + . Presented is SAR expression on the CD4 + NGFR + population. (G) Mean fluorescence intensity (MFI) of respective SARs on single and dual-receptor engineered T cells. Data are from 4 experiments with 4 PBMC donors (each donor is represented by a unique symbol). (H) Cytotoxicity of single, dual, and non-transduced T cell products after 120-h co-culture with U-251 tumor cells in an Incucyte assay. All conditions were normalized to growth of tumor cells alone. Data are from 4 individual experiments with 3 PBMC donors. Error bars represent SEM of technical replicates. (I) Cytotoxicity of single, dual, and non-transduced T cell products after 120-h co-culture with U-251 IL13Rα2 KO and U-251 HER2 KO cells in an IncuCyte assay. Data are representative of 3 experiments with 3 PBMC donors. Error bars represent SEM. Statistical analysis for (G) and (H) were performed using a paired t test and two-way ANOVA with correction for multiple comparison (Tukey test), respectively (∗ p = ≤ 0.05, ∗∗ p = ≤ 0.01, ∗∗∗ p = ≤ 0.001, ∗∗∗∗ p = ≤ 0.0001).

Article Snippet: Other phenotypic markers were detected with mouse anti-human CD4 AlexaFluor700 (Invitrogen), mouse anti-human CD8a PerCP-Cyanine5.5 (Invitrogen), mouse anti-human CD8 BV786 (BD Biosciences) and mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Expressing, Construct, Transduction, Binding Assay, Co-Culture Assay, Cell Counting, Flow Cytometry, Fluorescence, Comparison

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Combinatorial targeting of CD133 and HER2 can be achieved using the dual-SAR approach (A) Schematic diagram of cDNA encoding CD133-SAR constructs. (B) SAR surface expression on primary human T cells as determined by binding of a myc-tag specific mAb or HER2-Fc to detect the IL13Rα2-KIR and HER2-TREM1 SARs, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + . (C) T cells were incubated with firefly luciferase-expressing HCT-116 tumor cells for 20 h at indicated effector:target ratios. Luminescence was read with an open filter upon addition of 0.15 mg/mL D-luciferin substrate and converted to % cytotoxicity. Error bars display standard deviation for technical replicates. (D) T cells were CTV-labeled and incubated for 72-h with HCT-116 tumor cells at a 1:1 ratio. T cell proliferation was measured by flow cytometry with live > CD3 + > CD4 + > NGFR+ cells presented. (E) Schematic diagram of cDNA encoding single and dual CD133/HER2 SAR constructs. (F) SAR surface expression of single and dual CD133/HER2 SAR T cells determined by binding of a FLAG tag specific mAb or HER2-Fc to detect the CD133-NKp44 and HER2-TREM1 SARs, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + . Presented is SAR expression on the CD4 + NGFR + population. (G) Mean fluorescence intensity (MFI) of respective SARs on single and dual-receptor engineered T cells. Data are from 4 experiments with 4 PBMC donors (each donor is represented by a unique symbol). (H) Cytotoxicity of single, dual, and non-transduced T cell products after 72-h co-culture with HCT-116 tumor cells in an Incucyte assay. All conditions in were normalized to growth of tumor cells alone. Data are from 4 individual experiments with 3 PBMC donors. Error bars represent SEM. (I) Cytotoxicity of single, dual and non-transduced T cell products after 120-h co-culture with HCT-116 CD133 KO and HCT-116 HER2 KO cells in an IncuCyte assay. Data are representative of 3 experiments with 3 PBMC donors. Error bars represent SEM of technical replicates. Statistical analysis for (G) and (H) were performed using a paired t test and two-way ANOVA with correction for multiple comparison (Tukey test), respectively (∗ p = ≤ 0.05, ∗∗ p = ≤ 0.01, ∗∗∗ p = ≤ 0.001, ∗∗∗∗ p = ≤ 0.0001).

Article Snippet: Other phenotypic markers were detected with mouse anti-human CD4 AlexaFluor700 (Invitrogen), mouse anti-human CD8a PerCP-Cyanine5.5 (Invitrogen), mouse anti-human CD8 BV786 (BD Biosciences) and mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Construct, Expressing, Binding Assay, Incubation, Luciferase, Standard Deviation, Labeling, Flow Cytometry, FLAG-tag, Fluorescence, Co-Culture Assay, Comparison

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Expression of multiple receptors attenuates the production of inflammatory cytokines but not proliferation (A and D) Schematic representation of stimulation conditions for single- vs. dual-SAR engagement against (A) U-251 tumor cells or (D) HCT-116 tumor cells. (B and E) Intracellular cytokine production by (B) U-251 or (E) HCT-116 stimulated engineered T cells was measured by flow cytometry. Data are presented as percent NGFR+ CD4 and CD8 T cells producing respective cytokines. Data are from four independent experiments with 3 PBMC donors. Each donor is represented by a unique symbol. For the gating strategy see Figure S6 A. (C and F) Engineered T cells were labeled with CellTrace Violet (CTV) and stimulated with (C) U-251 or (F) HCT-116 tumor cells at a 1:1 ratio for 72 h. T cell proliferation and absolute cell count using 123count eBeads were measured by flow cytometry. Data are from three independent experiments with 2 T cell donors. Each donor is represented by a unique symbol. For the gating strategy see Figure S6 . Statistical analysis for (B), (C), (E), and (F) were performed using two-way ANOVA with correction for multiple comparison (Tukey test) (∗ p = ≤ 0.05, ∗∗ p = ≤ 0.01, ∗∗∗ p = ≤ 0.001, ∗∗∗∗ p = ≤ 0.0001, ns = not significant.

Article Snippet: Other phenotypic markers were detected with mouse anti-human CD4 AlexaFluor700 (Invitrogen), mouse anti-human CD8a PerCP-Cyanine5.5 (Invitrogen), mouse anti-human CD8 BV786 (BD Biosciences) and mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Expressing, Flow Cytometry, Labeling, Cell Counting, Comparison

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Expression of multiple receptors attenuates the early signal strength of individual receptors (A and B) Intracellular phospho-specific staining of ERK. Cells were stimulated for 30 min with respective WT or KO tumor lines, fixed, methanol-permeabilized and stained for phosphorylated ERK 1/2 (pT202/pY204). (A) Representative plots from 1 of 4 independent experiments and (B) %pERK+ of single and dual-SAR T cells following stimulation with a single target antigen. Data are from 4 independent experiments and 3 T cell donors. (C) Cells were stimulated with HCT-116 CD133 KO or U251 IL13Rα2 KO tumor cells for 1–4 h. The cells were surface stained for viability, CD4, CD8, NGFR and CD69, fixed/permeabilized, and then stained intracellularly/intranuclearly for Nur77. Cells were gated as follows: lymphocytes > single cells > live > CD4/CD8 > NGFR + > Nur77/CD69. Presented is Nur77 and CD69 expression on the CD8 + NGFR + population. Data are generated with T cells from one donor. For the gating strategy, see Figure S6 C.

Article Snippet: Other phenotypic markers were detected with mouse anti-human CD4 AlexaFluor700 (Invitrogen), mouse anti-human CD8a PerCP-Cyanine5.5 (Invitrogen), mouse anti-human CD8 BV786 (BD Biosciences) and mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Expressing, Staining, Generated

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet:

Article Snippet: Other phenotypic markers were detected with mouse anti-human CD4 AlexaFluor700 (Invitrogen), mouse anti-human CD8a PerCP-Cyanine5.5 (Invitrogen), mouse anti-human CD8 BV786 (BD Biosciences) and mouse anti-human NGFR (Miltenyi Biotec).

Techniques: FLAG-tag, Control, Virus, Generated, Subcloning, Recombinant, Staining, Flow Cytometry, Selection, Gene Knockout, Plasmid Preparation, Cloning, Expressing, Software

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet:

Article Snippet: Mouse anti-CD271 (NGFR), VioBright FITC (clone ME20.4-1.H4) , Miltenyi Biotec , Cat# 130-113-423, RRID: AB_2734064.

Techniques: FLAG-tag, Control, Virus, Generated, Subcloning, Recombinant, Staining, Flow Cytometry, Selection, Gene Knockout, Plasmid Preparation, Cloning, Expressing, Software