anti cd3  (Miltenyi Biotec)

Journal: iScience

Article Title: Uncovering the individual immunotherapeutic roles of PTPN1 and PTPN2 in T cells during dual inhibition

doi: 10.1016/j.isci.2025.113610

Figure Lengend Snippet: L598 is a PTPN1/2 dual inhibitor that enhances CTL responses against cancer cells (A) Molecular structure of L598, a phosphotyrosine mimetic inhibitor of PTPN1 and PTPN2. (B) Flow cytometric analysis of OT-1-induced cytotoxicity with increasing concentrations of L598 (2, 5, and 10 μM). E.G7 target cells were labeled with cell trace CFSE, and viability was assessed with the Life/Death UV450 staining. (C) Dot plot of the cytotoxicity induced by OT-1 cells at different effector-to-target ratios and concentrations of L598 (CD8 + :E.G7). (D) Flow cytometric analysis of naive (Tn, CD62L + , CD44 − ), central memory (Tcm, CD62L + , CD44 + ), and effector (Teff, CD62L − , CD44 + ) subpopulations gated on live CD8 + T cells after 4 days of expansion. (E) Stacked bar graph of central memory (Tcm) and effector subset (Teff) proportions within CD8 + T cells from (D). (F) Flow cytometric analysis of human naive (Tn, CD62L + , CD45RO − ), central memory (Tcm, CD62L + , CD45RO + ), and effector memory (Tem, CD62L − , CD45RO + ) subpopulations gated on live CD8 + T cells after 7 days of expansion. (G) Bar plot of the proportion of human CD8 Tcm (central memory) and Teff (effector) with increasing concentration of PTPN1/2 inhibitor L598 from (G). Data representative of n = 3 biological replicates (mice) for N2KO, N2KO/N1HET, dKO, and L598. Data representative of n = 2 biological replicates for N1KO (mice) and (G and H) human peripheral blood mononuclear cell donors. (H) Flow cytometric analysis of IFN-γ and TNF-α production in human T cells expanded and stimulated with anti-CD3/CD28. (I) Bar chart of the frequency of INF-γ- and TNF-α-positive T cells within the Tem (CD62L − , CD45RO + ) and Tcm (CD62L + , CD45RO + ) subsets. Data representative of two independent biological replicates/donors. Data representative of at least three independent experiments or biological replicates (mice). Error bars represent mean ± SEM. Statistical analysis: (C, E, G) two-way ANOVA with Dunnett’s multiple comparison test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: anti-human CD3 Antibody , Miltenyi Biotec , Cat# 130-093-377.

Techniques: Labeling, Staining, Concentration Assay, Comparison

Journal: Cell Reports Medicine

Article Title: Overcoming resistance to immunotherapy by targeting CD38 in human tumor explants

doi: 10.1016/j.xcrm.2025.102210

Figure Lengend Snippet: CD38 hi CD8 + T cells are dysfunctional (A) Expression of exhaustion and effector/memory-related genes in CD8 + TILs from human melanoma tumors. (B) Co-expression deviation proportion plot demonstrating co-expression of exhaustion-related genes and TCF7 from melanoma validation cohort. (C and D) differentially expressed genes based on CD38 expression in (C) CD8 + TILs from human melanoma and (D) CD3 + TILs from B16-ova murine melanoma. (E and F) CD38 hi and CD38 lo B7-H3.CAR-T cells (E) surface staining of PD-1 + CD39 + TIM-3 + ( n = 3; two-sided paired t test) and (F) TCF7 expression ( n = 3; two-sided unpaired t test). Means (bars) and individual values (open circles) are shown. (G) Scheme depicting acute and chronic TCR stimulation. (H–J) (H) Acute and chronic B7-H3.CAR-T proliferation assay ( n = 3; two-way ANOVA with Sidak correction for multiple comparisons). Means ± SEM (shaded area) are shown. Staining of (I) CD38 + CD39 + and (J) PD-1 + TIM-3 + ( n = 3, two-sided paired t test). Means (bars) and individual values (open circles) are shown. (K) Cytotoxicity assay toward 10164 patient-derived melanoma cell line. A representative experiment out of three is presented; two more are in E and S4F. ( n = 3 biological replicates; three independent experiments; two-way ANOVA with Sidak correction for multiple comparisons). Means ± SEM (shaded area) are shown. (L–N) Analysis of chronically stimulated control sgRNA and CD38 sgRNA B7-H3.CAR-T cells. (L) Proliferation assay ( n = 3 biological replicates; three independent experiments; two-way ANOVA with Sidak correction for multiple comparisons). Means ± SEM (shaded area) are shown. (M) TCF7 intracellular staining ( n = 4; two-sided paired t test). Means (bars) and individual values (open circles) are shown. (N) Cytotoxicity assay against 10164 melanoma cells ( n = 3 biological replicates; three independent experiments; two-way ANOVA with Sidak correction for multiple comparisons). Means ± SEM (shaded area) are shown. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. See also ; and .

Article Snippet: Peripheral blood mononuclear cells (PBMCs) isolated from whole blood were plated with CD3 (Miltenyi, 130-093-377) and CD28 (BD Bioscience, 556620) mAbs.

Techniques: Expressing, Biomarker Discovery, Staining, Proliferation Assay, Cytotoxicity Assay, Derivative Assay, Control

Journal: Cell Reports Medicine

Article Title: Overcoming resistance to immunotherapy by targeting CD38 in human tumor explants

doi: 10.1016/j.xcrm.2025.102210

Figure Lengend Snippet: CD38 + T cells exhibit altered bioenergetics (A) Correlation analysis between the GSEA of CD38 +/− CD8 + T cells in human melanoma and CD38 +/− CD3 + T cells in B16-ova murine melanoma. (B–E) Flow cytometry of (B and D) mitochondrial mass and (C and E) MMP in indicated groups ( n = 3; two-sided paired t test). Means (bars) and individual values (open circles) are shown. (F) Oxygen consumption rate (OCR) under basal condition and in response to inhibitors ( n = 5, two biological replicates, two-way ANOVA with Sidak correction for multiple comparisons). Data are presented as mean ± SEM. (G and H) Relative levels of (G) NAD + (nicotinamide adenine dinucleotide) and (H) NADP + (nicotinamide adenine dinucleotide phosphate); log 2 fold change (L2FC) from control is shown ( n = 6 biological replicates; two independent experiments; two-sided unpaired t test). Means (bars) and individual values (open circles) are shown. (I) scheme of NAD + metabolism and L2FC of indicated analytes. (J) OCR as in (F) of B7-H3.CAR-T cells ± CD38i ( n = 5, two biological replicates, two-way ANOVA with Sidak correction for multiple comparisons). Data are presented as mean ± SEM. (K and L) staining of (K) TIM-3 + PD-1 + and (L) CD39 + TIM-3 + in B7-H3.CAR-T ± CD38i ( n ≥ 4; two-sided paired t test). Means (bars) and individual values (open circles) are shown. (M) TCF7 expression by RT-qPCR in B7-H3.CAR-T cells in indicated groups ( n = 4; two-sided unpaired t test). Means (bars) and individual values (open circles) are shown. (N and O) Relative levels of (N) NAM (nicotinamide) and (O) ADPR (adenosine diphosphate ribose) in B7-H3.CAR-T cells ± CD38i ( n = 6 biological replicates; two independent experiments; two-sided unpaired t test). Means (bars) and individual values (open circles) are shown. ∗ p < 0.05, ∗∗ p < 0.01. See also ; , , and .

Article Snippet: Peripheral blood mononuclear cells (PBMCs) isolated from whole blood were plated with CD3 (Miltenyi, 130-093-377) and CD28 (BD Bioscience, 556620) mAbs.

Techniques: Flow Cytometry, Control, Staining, Expressing, Quantitative RT-PCR

Journal: Cell Reports Medicine

Article Title: Overcoming resistance to immunotherapy by targeting CD38 in human tumor explants

doi: 10.1016/j.xcrm.2025.102210

Figure Lengend Snippet: CD38 hi CD8 + T cells are dysfunctional (A) Expression of exhaustion and effector/memory-related genes in CD8 + TILs from human melanoma tumors. (B) Co-expression deviation proportion plot demonstrating co-expression of exhaustion-related genes and TCF7 from melanoma validation cohort. (C and D) differentially expressed genes based on CD38 expression in (C) CD8 + TILs from human melanoma and (D) CD3 + TILs from B16-ova murine melanoma. (E and F) CD38 hi and CD38 lo B7-H3.CAR-T cells (E) surface staining of PD-1 + CD39 + TIM-3 + ( n = 3; two-sided paired t test) and (F) TCF7 expression ( n = 3; two-sided unpaired t test). Means (bars) and individual values (open circles) are shown. (G) Scheme depicting acute and chronic TCR stimulation. (H–J) (H) Acute and chronic B7-H3.CAR-T proliferation assay ( n = 3; two-way ANOVA with Sidak correction for multiple comparisons). Means ± SEM (shaded area) are shown. Staining of (I) CD38 + CD39 + and (J) PD-1 + TIM-3 + ( n = 3, two-sided paired t test). Means (bars) and individual values (open circles) are shown. (K) Cytotoxicity assay toward 10164 patient-derived melanoma cell line. A representative experiment out of three is presented; two more are in E and S4F. ( n = 3 biological replicates; three independent experiments; two-way ANOVA with Sidak correction for multiple comparisons). Means ± SEM (shaded area) are shown. (L–N) Analysis of chronically stimulated control sgRNA and CD38 sgRNA B7-H3.CAR-T cells. (L) Proliferation assay ( n = 3 biological replicates; three independent experiments; two-way ANOVA with Sidak correction for multiple comparisons). Means ± SEM (shaded area) are shown. (M) TCF7 intracellular staining ( n = 4; two-sided paired t test). Means (bars) and individual values (open circles) are shown. (N) Cytotoxicity assay against 10164 melanoma cells ( n = 3 biological replicates; three independent experiments; two-way ANOVA with Sidak correction for multiple comparisons). Means ± SEM (shaded area) are shown. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. See also ; and .

Article Snippet: Anti-human CD3 , Miltenyi , Cat#130-093-377 RRID: N/A.

Techniques: Expressing, Biomarker Discovery, Staining, Proliferation Assay, Cytotoxicity Assay, Derivative Assay, Control

Journal: Cell Reports Medicine

Article Title: Overcoming resistance to immunotherapy by targeting CD38 in human tumor explants

doi: 10.1016/j.xcrm.2025.102210

Figure Lengend Snippet: CD38 + T cells exhibit altered bioenergetics (A) Correlation analysis between the GSEA of CD38 +/− CD8 + T cells in human melanoma and CD38 +/− CD3 + T cells in B16-ova murine melanoma. (B–E) Flow cytometry of (B and D) mitochondrial mass and (C and E) MMP in indicated groups ( n = 3; two-sided paired t test). Means (bars) and individual values (open circles) are shown. (F) Oxygen consumption rate (OCR) under basal condition and in response to inhibitors ( n = 5, two biological replicates, two-way ANOVA with Sidak correction for multiple comparisons). Data are presented as mean ± SEM. (G and H) Relative levels of (G) NAD + (nicotinamide adenine dinucleotide) and (H) NADP + (nicotinamide adenine dinucleotide phosphate); log 2 fold change (L2FC) from control is shown ( n = 6 biological replicates; two independent experiments; two-sided unpaired t test). Means (bars) and individual values (open circles) are shown. (I) scheme of NAD + metabolism and L2FC of indicated analytes. (J) OCR as in (F) of B7-H3.CAR-T cells ± CD38i ( n = 5, two biological replicates, two-way ANOVA with Sidak correction for multiple comparisons). Data are presented as mean ± SEM. (K and L) staining of (K) TIM-3 + PD-1 + and (L) CD39 + TIM-3 + in B7-H3.CAR-T ± CD38i ( n ≥ 4; two-sided paired t test). Means (bars) and individual values (open circles) are shown. (M) TCF7 expression by RT-qPCR in B7-H3.CAR-T cells in indicated groups ( n = 4; two-sided unpaired t test). Means (bars) and individual values (open circles) are shown. (N and O) Relative levels of (N) NAM (nicotinamide) and (O) ADPR (adenosine diphosphate ribose) in B7-H3.CAR-T cells ± CD38i ( n = 6 biological replicates; two independent experiments; two-sided unpaired t test). Means (bars) and individual values (open circles) are shown. ∗ p < 0.05, ∗∗ p < 0.01. See also ; , , and .

Article Snippet: Anti-human CD3 , Miltenyi , Cat#130-093-377 RRID: N/A.

Techniques: Flow Cytometry, Control, Staining, Expressing, Quantitative RT-PCR