t cell activation transacttm miltenyi biotec  (Miltenyi Biotec)

Journal: Nature Communications

Article Title: Glycosylated extracellular matrix drives immune suppression by modulating macrophage-T cell crosstalk in triple-negative breast cancer

doi: 10.1038/s41467-026-73467-5

Figure Lengend Snippet: a Representative images of TNBC patient tissue stained with anti-panCK (DAB) for tumor cells and anti-CD8 (VIP) for CD8+ cytotoxic T cells. Scale bar = 100 µm. Tumor and stroma annotations and CD8 T cell detections performed using QuPath as illustrated. Intensity features for annotations calculated using QuPath. N = 18 tissues. b Pearson’s r value for correlative analysis of CD8% in the stroma compared to CD8% in the tumor. Red = inflamed, green = excluded, blue = desert. N = 17 tissues. c Pie chart showing distribution of phenotypes for 17 tissues. ECM N-linked glycans assigned to four main groups (Complex, purple; Hybrid, dark red; High-mannose, red; Paucimannose, orange). Stacked barchart displaying the proportion of each family in the N-glycome for d , mean inflamed and excluded samples and individual e , inflamed or f , excluded samples. g – i ECM N-linked complex glycans assigned to four main groups (Non-Sialyl, blue; Mono-Sialyl, orange; Di-Sialyl, red; Tri-Sialyl, dark red; Tetra-Sialyl,, purple). Stacked barchart displaying the proportion of each family in the N-glycome for g , mean inflamed and excluded samples and individual h , inflamed or i , excluded samples. d – i Mean with SD. Two-way Repeated Measures ANOVA with Šídák’s multiple comparisons test, N = 4 excluded tissues, N = 3 inflamed tissues.

Article Snippet: Isolated T cells were activated for 24 h using T Cell TransActTM, human (130-128-758, Miltenyi) with 1:500 IL-2 (1 × 10 5 IU/mL, 200-02-100UG, ThermoFisher) according to manufacturer’s instructions.

Techniques: Staining

Journal: Nature Communications

Article Title: Glycosylated extracellular matrix drives immune suppression by modulating macrophage-T cell crosstalk in triple-negative breast cancer

doi: 10.1038/s41467-026-73467-5

Figure Lengend Snippet: a Schematic of a fluorescence-based live confocal imaging assay using decellularized tissue. Created in BioRender (Tarantola, L., 2026) https://BioRender.com/de0fus5 . b Representative images of adjacent and tumor decellularized tissue recellularized with mCherry HCC38 TNBC cells (red) and T cells (green) with ECM stained with anti-FN1-AF405 (blue). Scale bar = 100 µm. Total T cell number per field of view (FOV) ( c ) and T cell number near (<5 µm) or far (> 5 µm) from cancer cells ( d ) in tumor and adjacent tissues. N = 3 donors/group, 5–10 FOV/tissue. Error bars represent mean ± SD. Density distributions of T cell speed ( e ) and track length ( f ) near (<5 µm) cancer cells on tumor (red) and adjacent (orange) tissue. N = 3 tumor and N = 3 adjacent tissues, 2 technical replicates each. Density distributions of T cell speed ( g ) and track length ( h ) near (<5 µm) or far (>5 µm) from cancer cells in tumor tissue. N = 3 tumor tissues, 2 technical replicates each. c – h Dashed lines indicate medians. Data are presented in log scale. Kruskal–Wallis test with Dunn’s multiple comparisons test (two-sided, adjusted for multiple comparisons). i Representative images of tumor control tissue and tumor tissue treated with PNGase F or neuraminidase and recellularized with mCherry HCC38 TNBC cells (red) and T cells (green), with ECM stained with anti-FN1-AF405 (blue). Scale bar = 100 µm. Total T cell number per FOV ( j ), T cells near cancer cells (<5 µm) ( k ), and far from cancer cells (>5 µm) ( l ). N = 3 donors, 6–10 FOV/tissue. Error bars represent mean ± SD. Density distributions of T cell speed ( m ) and track length ( n ) near (<5 µm) cancer cells on tumor (red), PNGase F-treated (violet), or neuraminidase-treated (green) tissue. Dashed lines indicate medians. Data are presented in log scale. N = 3 tumor tissues, 2 technical replicates each. j – n Kruskal–Wallis test with Dunn’s multiple comparisons test (two-sided, adjusted for multiple comparisons).

Article Snippet: Isolated T cells were activated for 24 h using T Cell TransActTM, human (130-128-758, Miltenyi) with 1:500 IL-2 (1 × 10 5 IU/mL, 200-02-100UG, ThermoFisher) according to manufacturer’s instructions.

Techniques: Fluorescence, Imaging, Staining, Control

Journal: Nature Communications

Article Title: Glycosylated extracellular matrix drives immune suppression by modulating macrophage-T cell crosstalk in triple-negative breast cancer

doi: 10.1038/s41467-026-73467-5

Figure Lengend Snippet: a Schematic of macrophage decellularized tissue culture co-cultured with donor-matched T cells, with or without cancer cell (CC) media. Monocytes from three separate blood donors were cultured for 14 days on tumor ( N = 3) or adjacent (N = 3) decellularized tissues treated with neuraminidase (Neu) or sodium acetate vehicle control. Flow gating strategy provided in Supplementary Fig. . Created in BioRender. Tarantola, L. (2026) https://BioRender.com/ohvhmfy . Stacked bar of b activation markers (ICOS & CD137) or c exhaustion markers (PD1, LAG3, TIM3) shown as percentage positive cells expressing one marker, two markers or three markers from the T cell population. Error bars represent mean ± SD. 2way ANOVA test. N = 3 ( N = 3 adjacent, N = 3 tumor, N = 3 blood cones). d Barchart of flow cytometry expression patterns of PD1, LAG3 and TIGIT shown as percentage of positive cells from T cell population. Line at mean with standard deviation error bars Mixed-effects analysis with Tukey’s multiple comparisons adjustment test (two-sided). N = 3 ( N = 3 adjacent, N = 3 tumor, N = 3 blood cones). e Heatmap showing log10-transformed concentrations (pg/mL) of 8 cytokines (IFN-γ, TNF-α, IL-6, IL-10, IL-12p70, IL-17A, IL-18 and IL-23) measured in culture supernatants from macrophage (MAM) and T cell co-cultures grown on decellularized adjacent or tumor ECM scaffolds, in the absence or presence of cancer cell–conditioned medium (CC). ECM scaffolds were either untreated or treated with neuraminidase (Neu) prior to cell seeding. N = 3 ( N = 3 adjacent, N = 3 tumor, N = 3 blood cones). Overlaid numbers indicate paired t-test p-values for comparisons between untreated and Neu-treated ECM within the same tissue and CC condition. f Barchart of flow cytometry expression patterns of CD62L and CD162 shown as percentage of positive cells from T cell population. Line at mean with standard deviation error bars. Mixed-effects analysis with Tukey’s multiple comparisons adjustment test (two-sided). N = 3 ( N = 3 adjacent, N = 3 tumor, N = 3 blood cones).

Article Snippet: Isolated T cells were activated for 24 h using T Cell TransActTM, human (130-128-758, Miltenyi) with 1:500 IL-2 (1 × 10 5 IU/mL, 200-02-100UG, ThermoFisher) according to manufacturer’s instructions.

Techniques: Cell Culture, Control, Activation Assay, Expressing, Marker, Flow Cytometry, Standard Deviation, Transformation Assay

Journal: bioRxiv

Article Title: A Conserved Metabolic–Oxidative Axis Underlies Immune Cell Cryo-vulnerability

doi: 10.64898/2026.03.26.714376

Figure Lengend Snippet: (A) LY294002 (25 μM), Rapamycin (2 μM), 2-DG (8 mM), 4-OI (50 μM) and/or Rosi (50 μM)-treated activated αβ T cell recovery 24 h after thawing (n = 4 healthy donors). (B) 4-OI (50 μM) and Rosi (50 μM)-pretreated activated αβ T cell ADCC assay using anti-CD3/CD20 antibody. T cells were cultured with Raji cells at the 5:1, 10:1 and 20:1 effector:target ratio for 24 h (n = 3 healthy donors). (C) LY294002 (25 μM), Rapamycin (2 μM), 2-DG (10 mM), 4-OI (200 μM) and/or Rosi (50 μM)-treated activated macrophages recovery 24 h after thawing. (D) LY294002 (25 μM), Rapamycin (2 μM), 2-DG (8 mM), 4-OI (50 μM) and/or Rosi (50 μM)-treated activated γδ T cell recovery 24 h after thawing (n = 4 healthy donors). (E) 2-DG (8 mM), or 4-OI (50 μM) + Rosi (50 μM)-pretreated activated γδ cytotoxicity assay with K562 cells (n = 3 healthy donors). (F) Normalized survival of mice transplanted γδ T cell (n = 3 mice/group). (G) IFN-γ levels of blood in treated mice (n = 3 mice/group). (H) Schematic diagram for vivo experiment. (I) Tumor growth in mice. (J) Luminescent images of treated mice (n = 5 mice/group). Data are the mean ± SEM (A to G, and I). Statistical analysis was performed using an unpaired two-tailed Student’s t test. Ns, P >0.05, *P <0.05, **P <0.01, ***P <0.001. Cryo: cryopreserved cells; Non-Cryo: non-cryopreserved cells.

Article Snippet: Human αβ T cells were isolated and activated from human peripheral blood mononuclear cells (PBMCs) using the T Cell TransActTM kit (Miltenyi Biotec, #130-111-160), in strict accordance with the manufacturer’s protocol.

Techniques: Cell Recovery, ADCC Assay, Cell Culture, Cytotoxicity Assay, Two Tailed Test