human tgfβ1 duoset elisa kit  (R&D Systems)

Journal: Cell Reports Medicine

Article Title: CD4 + anti-TGF-β CAR T cells and CD8 + conventional CAR T cells exhibit synergistic antitumor effects

doi: 10.1016/j.xcrm.2025.102020

Figure Lengend Snippet: CD4 + but not CD8 + T28zT2 T cells are effective for tumor growth inhibition (A) CD4 + and CD8 + T cell compartments were sorted, and CD8 + T28zT2 T cells and CD4 + T28zT2 cells were generated. We also mixed the CD4 + and CD8 + T28zT2 T cells at a ratio of 1:1 to obtain CD3 + T28zT2 T cells. We then infused a total of 5 × 10 6 of the three types of T28zT2 T cells, CD3 + 1928zT2 T cells, or CD3 + M28zT2 T cells peritumorally into subcutaneous xenografts of NSI mice inoculated with 1 × 10 6 AsPc-1 cells (day 0). Tumor volumes were monitored on the indicated days ( n = 8 mice/T28zT2 CD3, T28zT2 CD4, and T28zT2 CD8 groups; n = 6 mice/1928zT2 CD3, M28zT2 CD3 and PBS groups); data are the mean ± SD values; two-way ANOVA with Tukey’s multiple comparisons test; ∗∗ p ≤ 0.01, ∗∗∗∗ p ≤ 0.0001. (B–D) CRISPR-Cas9-RNP knockout of GZMB or IFNG expression in CD4 + T28zT2 T cells. A total of 4 × 10 5 T28zT2 CD4 control single guide RNA (sgctrl), T28zT2 CD4 single guide RNA targeting granzyme B (sg GZMB ), T28zT2 CD4 single guide RNA targeting interferon-gamma (sg IFNG ), G28zT2 CD4 sgctrl, or 1928zT2 CD4 sgctrl T cells were cocultured with 1 × 10 5 Huh7 cells for 72 h. Shown are the levels of IFN-γ (B) and Granzyme B (C) (from 4 independent experiments) detected by ELISA assay; (D) the relative viability of Huh7 cells (from 4 independent experiments); data are the mean ± SEM values; one-way ANOVA with Tukey’s multiple comparisons test; ∗ p < 0.05, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. (E) Curves showing variations in the volume of Huh7 tumors in NSI mice post-infusion of T28zT2 CD4 sgctrl, T28zT2 CD4 sg IFNG , T28zT2 CD4 sg GZMB , G28zT2 CD4 sgctrl, or 1928zT2 CD4 sgctrl T cells ( n = 5 mice/group); data are the mean ± SD values; two-way ANOVA with Tukey’s multiple comparisons test; ∗∗∗∗ p ≤ 0.0001. See also in Figures S4 and .

Article Snippet: Human IFN-γ Precoated ELISA Kit , DAKEWE , Cat# 1110003.

Techniques: Inhibition, Generated, CRISPR, Knock-Out, Expressing, Control, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: CD4 + anti-TGF-β CAR T cells and CD8 + conventional CAR T cells exhibit synergistic antitumor effects

doi: 10.1016/j.xcrm.2025.102020

Figure Lengend Snippet: A combination of CD4 + anti-TGF-β CAR T cells and CD8 + anti-MSLN CAR T cells exhibits augmented antitumor effects in NSCLC PDX (A) NSCLC PDX tumors were diced into ∼30 mm 3 pieces, and tissue were inoculated subcutaneously into the right flanks of 8-week-old male NSI mice. 5 × 10 6 T4M828zT2, T28zT2 CD4, M28zT2, or 1928zT2 T cells were injected peritumorally (day 0). Tumor volumes were monitored on the indicated days ( n = 6 mice/group); data are the mean ± SD values; two-way ANOVA with Tukey’s multiple comparisons test; ∗∗ p ≤ 0.01, ∗∗∗∗ p ≤ 0.0001. (B–D) The mean fluorescence intensity (MFI) of PD-1 among tumor-infiltrating CAR + CD4 + (B, left) and CAR + CD8 + (B, right) T cells from the T4M828zT2 and M28zT2 groups on day 21 determined by flow cytometry ( n = 4 mice/group). (C and D) Data are shown as the mean ± SD values; unpaired two-tailed t test; ∗ p < 0.05, ∗∗ p ≤ 0.01. (E) A schematic diagram of the experimental design. Tumor tissue from 1928zT2, M28zT2, or T4M828zT2 groups was obtained from NSCLC PDX models at the endpoint (day 21). Tumor tissues were prepared into single-cell suspension. Tumor-infiltrating CAR + (GFP + ) CD8 + T cells from the M28zT2 and T4M828zT2 groups and tumor-infiltrating CD8 + T cells from the 1928zT2 group were sorted by FACS. These tumor-infiltrating T cells were then stimulated with CD3/CD28 monoclonal antibodies (mAbs) and subjected to functional experiments. Finally, the cytotoxicity, cytokine production, and T cell expansion of tumor-infiltrating CD8 + T cells from the 1928zT2, M28zT2, and T4M828zT2 groups were evaluated. Graphics were created with BioRender.com (agreement number QW27PPO9DF ). (F–I) Tumor-infiltrating CD8 + 1928zT2, CAR + CD8 + M28zT2, or CAR + CD8 + T4M828zT2 cells were incubated with AsPc-1 cells at a 2:1 effector (E):target (T) ratio for 72 h. (F) Representative images of 0.1% crystal violet staining of AsPc-1 cells cocultured with CD8 + 1928zT2, CAR + CD8 + M28zT2, or CAR + CD8 + T4M828zT2 tumor-infiltrating T cells ex vitro . (G) The relative viability of AsPc-1 cells with 1928zT2, M28zT2, or T4M828zT2 T-cell induced lysis after 72 h n = 4 mice/group. (H and I) Supernatants were harvested and analyzed with a multiplex immunoassay to determine the concentrations of the indicated cytokines. n = 4 mice/group. The concentrations of IFN-γ (H) and Granzyme B (I) were measured by ELISA assay; data are the mean ± SD values; one-way ANOVA with Tukey’s multiple comparisons test; ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. (J) The expansion of tumor-infiltrating CD8 + 1928zT2, CAR + CD8 + M28zT2, and CAR + CD8 + T4M828zT2 cells was detected by flow cytometry on day 0, 3 and 7; data are the mean ± SD values; n = 4 mice/group; two-way ANOVA with Tukey’s multiple comparisons test; ∗∗∗∗ p ≤ 0.0001. See also in Figure S6 .

Article Snippet: Human IFN-γ Precoated ELISA Kit , DAKEWE , Cat# 1110003.

Techniques: Injection, Fluorescence, Flow Cytometry, Two Tailed Test, Suspension, Bioprocessing, Functional Assay, Incubation, Staining, Lysis, Multiplex Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: CD4 + anti-TGF-β CAR T cells and CD8 + conventional CAR T cells exhibit synergistic antitumor effects

doi: 10.1016/j.xcrm.2025.102020

Figure Lengend Snippet:

Article Snippet: Human IFN-γ Precoated ELISA Kit , DAKEWE , Cat# 1110003.

Techniques: Purification, Functional Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Cell Isolation, Western Blot, Software

Journal: Frontiers in Immunology

Article Title: Targeting TGFβ with chimeric switch receptor and secreted trap to improve T cells anti-tumor activity

doi: 10.3389/fimmu.2024.1460266

Figure Lengend Snippet: Design and expression of TGFβR-based CSRs and anti-TGFβ trap. (A) Schematic representation of the concept: central panel depicts the CSR approach, right panel the trap approach, compared to left panel which shows native state (generated with BioRender). (B) Schematic representation of CSRs and trap encoding constructs used in this study. (EC – extracellular domain, TM – transmembrane domain, IC – intracellular domain, IRES – Internal ribosome entry site) (C) T cells were transduced with retroviral constructs encoding the different CSR or trap constructs followed by an IRES-NGFR sequence, or the control gene only (NGFR). Cells were analyzed for transduction efficiency by flow cytometer following staining for NGFR expression. Representative histograms with the percentage of positive cells and mean fluorescent intensity (MFI - in bracket) are shown. (D) Boxplot summary of the transduction efficiency with the different constructs as indicated (n=15, with 15 different donors; bars represent SEM). (E, F) Similarly, following transduction with the F4 TCR, T cells were analyzed for Vβ12 expression: (E) representative histograms from one experiment and (F) a boxplot summary of the results of n=15 independent experiments (with 15 different donors). No significant difference in F4 TCR expression by T cells transduced with constructs was found (p=0.32, calculated using ANOVA test; n=15, with 15 different donors). (G) T cells transduced with the Trap-IRES-NGFR constructs were stained for NGFR expression and using an His-tag specific antibody to detect trap expression. Representative histograms of NGFR expression and of intracellular staining of His tag are shown on the left panel and a representative dot-plot of both staining is shown on the right panel. These results are representative of n=3 experiments with 3 different donors. (H) Trap secretion was evaluated following stimulation of transduced cells with plate bound OKT3 at the indicated concentrations ranging from 0 to 10 ng/ml. Trap secretion in the medium was measured by ELISA, using HRP-labeled anti-His tag. These results are presented as mean ± SEM of n=3 independent experiments with 3 different donors. The differences between trap and w/o (no transduction) were found statistically significant (*p<0.002, calculated using Student’s t-test). (I) Binding ability of trap to TGFβ. Supernatant collected from lymphocytes, transduced with trap or not (w/o), was incubated in plates previously coated with TGFβ (+) or not (-). ELISA was performed using a His-tag antibody to detect trap binding. The results are the mean ± SEM of n=4 independent experiments with 4 different donors. The difference between TGFβ trap and w/o control was found to be statistically significant, as the difference between TGFβ coated and non-coated plates (*p<0.05, calculated using a paired Student’s t test).

Article Snippet: Melanoma cancer cell lines were tested for TGFβ secretion using a commercially available ELISA kit for human/mouse TGFβ1 (Invitrogen, ThermoFisher Scientific, Massachusetts, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Generated, Construct, Transduction, Retroviral, Sequencing, Control, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Labeling, Binding Assay, Incubation

Journal: Frontiers in Immunology

Article Title: Targeting TGFβ with chimeric switch receptor and secreted trap to improve T cells anti-tumor activity

doi: 10.3389/fimmu.2024.1460266

Figure Lengend Snippet: TGFβ and MART-1 antigen expression by melanoma cells lines. (A) Culture medium from melanoma cell lines as indicated was collected to determine TGFβ concentration using ELISA. The results are presented as mean ± SEM of n=3 independent experiments, the means difference found to be significantly different (p=0.07*10 -9 , calculated using ANOVA). (B) SK-MEL23 cell line was engineered to express mCherry, followed by transduction with TGFβ sequence-bearing vector to enhance TGFβ secretion. Supernatant from SK-MEL23 mCherry and SK-MEL23 mCherry/TGFβ cultures was evaluated for TGFβ content using ELISA. The results are presented as mean + SEM and the difference between the parental and engineered cell line was found to be statistically significant (*p=3*10 -5 , calculated using Student’s t-test). (C) Melanoma cells lines were analyzed using flow cytometry for HLA-A2 expression, essential for F4 TCR-dependent T cells activation. Representative histograms show the percentage of positive cells and mean fluorescent intensity (MFI in bracket). (D) For MART-1 antigen expression detection, total RNA was extracted from melanoma cell lines, followed by mRNA conversion to cDNA. The antigen expression was determined using PCR amplification with primers specific to a 365bp part of a gene, where cDNA was used as a template (NTC - No Template Control).

Article Snippet: Melanoma cancer cell lines were tested for TGFβ secretion using a commercially available ELISA kit for human/mouse TGFβ1 (Invitrogen, ThermoFisher Scientific, Massachusetts, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transduction, Sequencing, Plasmid Preparation, Flow Cytometry, Activation Assay, Amplification, Control

Journal: Frontiers in Immunology

Article Title: Targeting TGFβ with chimeric switch receptor and secreted trap to improve T cells anti-tumor activity

doi: 10.3389/fimmu.2024.1460266

Figure Lengend Snippet: CSRI and anti-TGFβ trap enhance T cells pro-inflammatory cytokine secretion. (A–C) Human primary lymphocytes were transduced to express the F4 TCR along with CSRs, trap or NGFR only (control); w/o represents mock transduced lymphocytes. Transduced cells were co-cultured with melanoma cell lines SK-MEL23, 938A2 and A375 (control) along with TGFβ (1.2 ng/ml) or not. IFNγ (A) , TNFα (B) and IL2 (C) secreted to the co-culture media was measured by ELISA. The results are presented as mean ± SEM, normalized to NGFR F4 (control) (n=7 with 7 different lymphocytes donors, *p ≤ 0.05, calculated using a paired Student’s t test). (D) Human primary lymphocytes transduced with trap were cultured for 48 h and their medium (CM – conditioned medium) was isolated to evaluate the function of the secreted trap. In parallel, human primary lymphocytes were transduced to express F4 TCR or not (w/o – without, represents mock-transduced control). Following transduction, T cells were co-cultured with melanoma cell line SK-MEL23 and A375 (control) along with TGFβ (0.4 -1.2 ng/ml) or not (0 ng/ml) in the trap containing medium (CM) isolated as aforementioned. Following the co-culture, IFNγ (D) , TNFα (E) and IL2 (F) secretion in the co-culture was measured using ELISA. The results were normalized to F4 TCR-transduced T cells co-cultured in CM of mock-transduced T cells without TGFβ. The results are presented as mean ± SEM (n=4 with 4 different donors, *p<0.05, n.s., not significant, calculated using a paired Student’s t test).

Article Snippet: Melanoma cancer cell lines were tested for TGFβ secretion using a commercially available ELISA kit for human/mouse TGFβ1 (Invitrogen, ThermoFisher Scientific, Massachusetts, USA), according to the manufacturer’s instructions.

Techniques: Control, Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Transduction, Isolation

Journal: Frontiers in Immunology

Article Title: Targeting TGFβ with chimeric switch receptor and secreted trap to improve T cells anti-tumor activity

doi: 10.3389/fimmu.2024.1460266

Figure Lengend Snippet: Phenotypic characterization of engineered T cells. (A–D) T cells were transduced with F4 TCR and CSRs, trap or NGFR only (control) and then co-cultured with SK-MEL23 with 1.2ng/ml TGFβ (+) or without (-). After the incubation, co-cultured lymphocytes were stained and analyzed by FACS, to detect the expression of 4-1BB (A) , CD69 (B) and IL2Rα (C) gated on the CD8 + population and of OX40 (D) , gated on the CD4 + population. These results were normalized to the expression observed in the NGFR F4 (control) population, co-cultured with SK-MEL23 without additional TGFβ (-). Results are presented as mean ± SEM (n=4 with 4 different donors, *p<0.05 calculated using a paired Student’s t test). (E) Following transduction, T cells were stained to determine CD4 + /CD8 + distribution by flow cytometry. The results are presented as mean ± SEM of n=6, with 6 different donors and no significant difference was detected (p>0.1, calculated using Student’s t-test). (F) Memory phenotype was also assessed by staining with CD45RO and CCR7, followed flow cytometry to detect the percentage of naïve, central memory (TCM), effector memory (TEM) and terminally differentiated effector memory (TEMRA) T cells in the transduced lymphocytes population. The results are presented as mean ± SEM of n=5, with 5 different donors. No statistically significant difference was detected (p>0.1, calculated using Student’s t-test). (G–I) T cells transduced with F4 TCR and CSRs, trap or NGFR (Control) were repeatedly co-cultured at an E:T ratio of 10:1 with parental melanoma line SK-MEL23 mCherry (-) or its TGFβ-transduced version (+). Every 2 days for a total of 8 days, fresh tumor targets were added to the co-cultures (total of 4 instances). At day 8, T cells were stained for the expression PD1 (G) , TIM3 (H) or TIGIT (I) , gated on the CD8 + population and analyzed by flow cytometer. Results are presented as mean ± SEM and normalized to that of NGFR F4 co-cultured with SK-MEL23 mcherry (-) (for (G) n=11, with 11 different donors and for (H, I) , n=6. with 6 different donors and *p<0.05, calculated using a paired Student’s t test).

Article Snippet: Melanoma cancer cell lines were tested for TGFβ secretion using a commercially available ELISA kit for human/mouse TGFβ1 (Invitrogen, ThermoFisher Scientific, Massachusetts, USA), according to the manufacturer’s instructions.

Techniques: Transduction, Control, Cell Culture, Incubation, Staining, Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Targeting TGFβ with chimeric switch receptor and secreted trap to improve T cells anti-tumor activity

doi: 10.3389/fimmu.2024.1460266

Figure Lengend Snippet: CSRI and anti-TGFβ trap can contribute to anti-tumor cytotoxicity in vitro and in a xenograft model. (A) Transduced T cells, expressing F4 TCR and CSR, trap or NGFR (control) were co-cultured with TGFβ-transduced SK-MEL23 mCherry target cells. Co-culture was performed at different E:T ratios as indicated. Fluorescent signal (OCU x µm²/Image) of live tumor cells (and normalized to t=0) was measured by Incucyte, every 2 h for 24h. Cytotoxicity was calculated as 100% - live cells %. Results at different E:T ratios are presented as mean ± SEM, n=6, with 6 different donors, and *p ≤ 0.05, calculated using a paired Student’s t test. (B) Additionally, these T cells were analyzed by flow cytometry for expression of CD107 degranulation marker after a short co-culture (2 h) with SK-MEL23 tumors targets in the presence (+) or not (-) of TGFβ. The results were normalized to that of NGFR F4 (control) co-cultured with SK-MEL23 without additional TGFβ (-) and are presented as mean ± SEM (n=5, with 5 different donors, *p<0.05 calculated using a paired Student’s t test). (C) T cells expressing F4 TCR and CSR, trap or NGFR were evaluated for their in vivo function. Briefly, NOD/SCID/Gamma mice were inoculated with 2 x 10 6 TGFβ-transduced SK-MEL23 cells along with 2 x 10 6 engineered lymphocytes in Cultrex. Tumor size (mm 3 ) was measured using a caliper and tumor size calculated using following formula: D x d 2 x π/6, where D is the largest tumor diameter and d is a perpendicular one. Results are presented as mean ± SEM (n=5, *p<0.05, calculated using Student’s t-test). The criteria for anti-tumor efficacy were based upon a significant delay in tumor development in treated mice, compared to NGFR positive control. (D) Mice survival was also evaluated and presented as a Kaplan-Meier plot, (with *p<0.05, calculated using LogRank test).

Article Snippet: Melanoma cancer cell lines were tested for TGFβ secretion using a commercially available ELISA kit for human/mouse TGFβ1 (Invitrogen, ThermoFisher Scientific, Massachusetts, USA), according to the manufacturer’s instructions.

Techniques: In Vitro, Expressing, Control, Cell Culture, Co-Culture Assay, Flow Cytometry, Marker, In Vivo, Positive Control

Journal: Cancer Research

Article Title: Human 3D Ovarian Cancer Models Reveal Malignant Cell–Intrinsic and –Extrinsic Factors That Influence CAR T-cell Activity

doi: 10.1158/0008-5472.CAN-23-3007

Figure Lengend Snippet: Inhibition of TGFβ signaling reduced ECM density and stimulated CAR T-cell activity in G164/FB gels. A, M3C, COL1A1, and FN1 staining and quantification of G164/FB gels treated with TGFβR inhibitor (TGFβR inhi). Scale bar, 50 μm. B, TWOMBLI analysis showing the heatmaps and %HDM for M3C, COL1A1, and fibronectin (FN1) in G164/FB gels treated with TGFβR inhibitor. Scale bar, 100 μm. C, Number of CD3 + CAR T cells at the peripheral and core regions of G164/FB collagen gels treated with TGFβR inhibitor. Two different CAR T-cell and FB donors were used for this experiment. D, Heatmaps generated using Definiens Tissue Studio showing the distribution of CAR T cells in G164/FB collagen gels treated with TGFβR inhibitor. Representative images from two/three gels per two replicates. Scale bar, 500 μm. E, IHC staining and quantification for caspase-3 (Casp3) on G164/FB gels treated with TGFβR inhibitor. Scale bar, 50 μm. F, Expression of FAP on G164/FB gels treated with TGFβR inhibitor. Scale bar, 50 μm. G, Behavior of CAR T cells in G164/FB gels treated with TGFβR inhibitor. Representative images from three repeats. Blue, FN1; green, CAR T cells; red, EpCAM. Scale bar, 50 μm. Speed and length of movement of migrating (vehicle, 83; TGFβR inhibitor, 62) CAR T cells. H, Number of CD3 + (left) and Casp3 + (right) on G164/FB gels treated with TGFβR inhibitor and aCCL2. A–C , E , F , and H, Data plotted as mean ± SD of two/three gels per two replicates. Statistics performed using unpaired t test ( A , B , and F ) and two-way ANOVA ( C , E , and H ).

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) was performed using human IFNα Quantikine kit (R&D Systems, Cat. DIF50C), human TNFα QuantiGlo kit (R&D Systems, Cat. QTA00C), human CCL2/MCP1 Quantikine kit (R&D Systems, Cat. DCP00), and human TGFβ1 Quantikine kit (R&D Systems, Cat. DB100C) according to manufacturer’s instructions.

Techniques: Inhibition, Activity Assay, Staining, Generated, Immunohistochemistry, Expressing

Journal: Endocrine

Article Title: Thyroid dysfunction in Hashimoto’s thyroiditis: a pilot study on the putative role of miR-29a and TGFβ1

doi: 10.1007/s12020-024-03965-3

Figure Lengend Snippet: Serum TGFβ1 levels in patients with normal thyroid function (Eu-HT), non-treated hypothyroidism (Hypo-HT) and hypothyroidism treated with replacement therapy LT4 (1.13 µg/kg) (subst-HT). TGFβ1 levels are reported as pg/mL (mean ± SD). **P < 0.01

Article Snippet: Serum TGF-β1 levels were detected by using Human TGFβ1 (Transforming Growth Factor Beta 1) ELISA Kit (EH0287, FineTest), according to the manufacturer’s protocol.

Techniques:

Journal: Endocrine

Article Title: Thyroid dysfunction in Hashimoto’s thyroiditis: a pilot study on the putative role of miR-29a and TGFβ1

doi: 10.1007/s12020-024-03965-3

Figure Lengend Snippet: Pearson correlation analysis of serum miR-29a(2^ −ΔΔCt ) with TGFβ1 levels (pg/mL); r = −0.75, P < 0.01

Article Snippet: Serum TGF-β1 levels were detected by using Human TGFβ1 (Transforming Growth Factor Beta 1) ELISA Kit (EH0287, FineTest), according to the manufacturer’s protocol.

Techniques: