Journal: iScience

Article Title: Cholinotrophic basal forebrain connectome dysfunction in Down syndrome with and without dementia

doi: 10.1016/j.isci.2025.113041

Figure Lengend Snippet: Images and quantitation of cholinotrophic basal forebrain neuron pathology within the nucleus basalis of Meynert in DSD+, DSD- and AMC cases (A–N) Photomicrographs show p75 NTR (brown, A-C), ChAT (purple, D-F) positive cells in AMC, DSD-, and DSD+ cases. Note the decrease in the number and intensity of both p75 NTR and ChAT labeled neurons in DSD- cases compared to an even greater reduction in DSD+ and globose shaped cells (black arrows) in DSD+ cases (C, F). Arrows in (A–F) mark cells shown at a higher magnification in the boxed areas located at the lower right corner of each panel. Dark brown AT8 (G, I) and TauC3 (H, J) bearing neurofibrillary tangles (NFTs) were observed only in the DSD- and DSD+ cases. Black arrows indicate globose shaped NFTs (G, H, and I) that are shown at a higher magnification in boxed areas adjacent to the lower magnification images. Note that not all neurons within the nbM (thin black arrows) contained tau pathology (G, H) in DSD- cases. Moreover, TauC3 staining revealed two NFT phenotypes that displayed either peripherally located or intense labeling that filled the entire structure (see boxed images adjacent to (H) and (J)). Scale bar in F = 50 μm and inset = 20 μm applies to panels A-E. Scale bar in J = 20 μm and inset = 20 μm applies to (G–J). Histograms show a significant reduction in both p75 NTR (K) and ChAT (L) positive cells in DSD+ compared to AMC. Although no significant were found in number of AT8 or TauC3 NFT positive cells (M), there was an increase in NTs in DSD+ compared to DSD- (N). ACM n = 5, DSD- n = 5, DSD+ n = 10. Data shown are presented as mean ± SEM. Statistical significance was determined using the Kruskal–Wallis’s test followed by Dunn’s test for comparisons across three clinical groups, and the Mann–Whitney test for comparisons between two groups (DSD- vs. DSD+). Significance levels (∗) were set at: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Antibodies were added to blocking buffer and membranes incubated overnight (4°C) in mouse anti-p75 NTR ([1:150], Santa Cruz Biotechnology Cat# sc-271708, RRID: AB_10714958 ), rabbit anti-ChAT ([1:500] (Proteintech Cat# 20747-1-AP, RRID: AB_10898169 ), rabbit anti-TrkA ([1:150], Fitzgerald Industries International Cat# 20R-TR013, RRID: AB_1289098 ) or rabbit anti-proNGF ([1:250], Alomone Labs Cat# ANT-005, RRID: AB_2040021 ), washed, incubated with horseradish peroxidase-conjugated with either a goat anti-mouse IgG (1:200) or goat anti-rabbit IgG (1:200) secondary antibody at RT, visualized by chemiluminescence (Kodak Image Station 440CF; Perkin-Elmer, Wellesley, MA), and quantified with Kodak 1.

Techniques: Quantitation Assay, Labeling, Staining, MANN-WHITNEY

Journal: iScience

Article Title: Cholinotrophic basal forebrain connectome dysfunction in Down syndrome with and without dementia

doi: 10.1016/j.isci.2025.113041

Figure Lengend Snippet: Immunofluorescent images and quantification of p75 NTR neurons with and without AT8 and MAP2, as well as Thioflavin S single and AT8/Thioflavin S dual-labeled nbM cells in DSD+ and DSD-cases. (A–U) Immunofluorescent neurons labeled with p75 NTR (A, E, green), MAP2 (B, F, red), AT8 (C, G, Cyan), ThS (blue) (I, L) and merge images (D, H, K, N, R) in DSD- and DSD+ cases. In the DSD-cases there were greater p75 NTR cells AT8 negative neurons and p75 NTR MAP2 dual labeled neurons (A-D) compared to the DSD+ cases (E–H). Note that not all p75 NTR cells colocalize with AT8 or MAP2 in both DS groups (D, H). To determine the stage of a tangle tissue was stained for ThS, a marker of advanced pathology and the early stage AT8 phosphorylation antibody. Note that there are only a few ThS-labeled tangles that displayed AT8 in both DS groups (yellow arrows), compared to single ThS tangles, which were greater in DSD+ than in DSD- (white arrows) (I-L). Note that ThS positive NFTs that do not contain AT8 also do not colocalize with MAP2 (O-R). Scale bar in F = 25 μm applies to panels A-G, N = 25 μm applies to panels I-M and R = 10 μm and applies to O-Q. Graph showing a significant reduction in both p75 NTR AT8 immuno-negative, and p75 NTR MAP2 dual labeled neurons in DSD+ compared to DSD- (S). ThS-positive cells were greater (T), while the percentage of double-labeled cells with AT8 and ThS decreased (U) in DSD+ compared to individuals without dementia. DSD- n = 5, DSD+ n = 5. Data are presented as mean ± SEM. Statistical significance was determined using Mann–Whitney test for comparisons between DSD- and DSD+. Significance levels (∗) were set at: ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Antibodies were added to blocking buffer and membranes incubated overnight (4°C) in mouse anti-p75 NTR ([1:150], Santa Cruz Biotechnology Cat# sc-271708, RRID: AB_10714958 ), rabbit anti-ChAT ([1:500] (Proteintech Cat# 20747-1-AP, RRID: AB_10898169 ), rabbit anti-TrkA ([1:150], Fitzgerald Industries International Cat# 20R-TR013, RRID: AB_1289098 ) or rabbit anti-proNGF ([1:250], Alomone Labs Cat# ANT-005, RRID: AB_2040021 ), washed, incubated with horseradish peroxidase-conjugated with either a goat anti-mouse IgG (1:200) or goat anti-rabbit IgG (1:200) secondary antibody at RT, visualized by chemiluminescence (Kodak Image Station 440CF; Perkin-Elmer, Wellesley, MA), and quantified with Kodak 1.

Techniques: Labeling, Staining, Marker, Phospho-proteomics, MANN-WHITNEY

Journal: iScience

Article Title: Cholinotrophic basal forebrain connectome dysfunction in Down syndrome with and without dementia

doi: 10.1016/j.isci.2025.113041

Figure Lengend Snippet: FC cholinotrophic protein levels in AMC, DSD- and DSD+ (A–D) Representative immunoblots, and bar graphs show a significant upregulation of (A) proNGF and (B) p75 NTR , while (C) ChAT protein was downregulated between AMC and DSD+. (D) TrkA protein levels were stable across the groups analyzed. ACM n = 5, DSD- n = 5, DSD+ n = 13. Data are presented as mean ± SEM. Statistical significance was determined using the Kruskal–Wallis’s test followed by Dunn’s test for comparisons across three clinical groups. Significance levels (∗) were set at: ∗ p < 0.05.

Article Snippet: Antibodies were added to blocking buffer and membranes incubated overnight (4°C) in mouse anti-p75 NTR ([1:150], Santa Cruz Biotechnology Cat# sc-271708, RRID: AB_10714958 ), rabbit anti-ChAT ([1:500] (Proteintech Cat# 20747-1-AP, RRID: AB_10898169 ), rabbit anti-TrkA ([1:150], Fitzgerald Industries International Cat# 20R-TR013, RRID: AB_1289098 ) or rabbit anti-proNGF ([1:250], Alomone Labs Cat# ANT-005, RRID: AB_2040021 ), washed, incubated with horseradish peroxidase-conjugated with either a goat anti-mouse IgG (1:200) or goat anti-rabbit IgG (1:200) secondary antibody at RT, visualized by chemiluminescence (Kodak Image Station 440CF; Perkin-Elmer, Wellesley, MA), and quantified with Kodak 1.

Techniques: Western Blot

Journal: iScience

Article Title: Cholinotrophic basal forebrain connectome dysfunction in Down syndrome with and without dementia

doi: 10.1016/j.isci.2025.113041

Figure Lengend Snippet: Summary of changes in the cholinotrophic basal forebrain connectome in DS Diagrammatic sagittal view of the human brain (A) and a modified stacked bar graph (B) illustrating differences in the pathobiology of the cholinotrophic projection system between non-trisomy age-matched control (AMC), DS without dementia (DSD-) and DS with dementia (DSD+) individuals with DS. Frontal cortex protein levels for ChAT (pink), proNGF (green), p75 NTR (purple) and TrkA (light blue). Nucleus basalis NFTs of ThS (blue), AT8 (orange) and TauC3 (black) and counts of p75 NTR and ChAT (red) neurons. Created with BioRender.com .

Article Snippet: Antibodies were added to blocking buffer and membranes incubated overnight (4°C) in mouse anti-p75 NTR ([1:150], Santa Cruz Biotechnology Cat# sc-271708, RRID: AB_10714958 ), rabbit anti-ChAT ([1:500] (Proteintech Cat# 20747-1-AP, RRID: AB_10898169 ), rabbit anti-TrkA ([1:150], Fitzgerald Industries International Cat# 20R-TR013, RRID: AB_1289098 ) or rabbit anti-proNGF ([1:250], Alomone Labs Cat# ANT-005, RRID: AB_2040021 ), washed, incubated with horseradish peroxidase-conjugated with either a goat anti-mouse IgG (1:200) or goat anti-rabbit IgG (1:200) secondary antibody at RT, visualized by chemiluminescence (Kodak Image Station 440CF; Perkin-Elmer, Wellesley, MA), and quantified with Kodak 1.

Techniques: Modification, Control

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