dose anti cd3 cd28 beads  (Miltenyi Biotec)

Journal: Cancer research

Article Title: Targeting the αvβ5 Integrin Modifies the TGF-β-Rich Tumor Microenvironment of Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-25-4223

Figure Lengend Snippet: A, Representative confocal images of Tregs in human PDAC and spleen specimens collected from an identical patient. The dotted lines outline cancer ducts. Red, CD3; white, Foxp3; green, αvβ5 integrin; blue, DAPI. Arrowheads, αvβ5 integrin + Tregs. The boxed area is magnified. Scale bars, 20 μm (large panels); 10 μm (small panels). The bar diagram summarizes the average number of αvβ5 + Tregs in the PDAC and spleen from 5 patients. The cells were counted in 5 randomly chosen HPFs per section. B, Flow cytometry analysis of naïve human CD4 + T cells before (left) and after (right) a 3-day-treatment with anti-CD3/CD28 Abs and TGF-β1. Note the induction of CD25 + Foxp3 + iTregs (red) that express αvβ5 integrin. CD25 − Foxp3 − non-Tregs (blue) remain negative for the integrin. Three biological replicates per group were analyzed. C, Representative microscopic images of αvβ5 integrin (blue) expressed in one of the five human PDAC specimens analyzed. Red box, with cancer ducts; green box, without cancer ducts. Black arrowhead, cancer duct; white arrowhead, stroma cell. Scale bars, 500 μm (left); 50 μm (middle); 20 μm (right). Statistical analysis, Student’s t test. Error bars, mean ± standard error.

Article Snippet: The CD4 + T cells were cultured for 3 days with or without KPC78 cells in the presence of low dose anti-CD3/CD28 beads (x40 dilution; Cat# 130-095-925; Miltenyi Biotec), recombinant mouse TGF-β1 (5 ng/ml; Cat# 7666-MB; R&D Systems, Minneapolis, MN), and recombinant mouse interleukin-2 (mIL-2, 100 U/ml; Cat# 11271164001; Roche, Basel, Switzerland), and subjected to flow cytometry as described elsewhere.

Techniques: Flow Cytometry

Journal: Cancer Research Communications

Article Title: Collagen-Bearing Exosomes from Breast Cancer–Associated Fibroblasts Promote T-cell Dysfunction

doi: 10.1158/2767-9764.CRC-25-0259

Figure Lengend Snippet: CAF exosomes directly inhibit T-cell proliferation. A, Schematic of experimental design. B and C, Proliferation of CFSE-labeled murine T cells 72 hours after concomitant stimulation (Stim.) with anti-CD3/CD28 antibodies and 10 μg/1 × 10 5 cells cancer cell or CAF exosomes (Exo.) from the MCaP line. Unstimulated (Unstim.) T cells used as a negative control. Dot plot of CD8 T cells shown in B . Proliferation of gated CD8 and CD4 T cells shown in C . D, Representative contour plot of granzyme B+ CD8 + T cells in the presence of anti-CD3/CD28 antibodies and cancer cell or CAF exosomes from the MCaP line for 72 hours. Quantification shown in E . F, Cancer cell or CAF exosomes (10 μg/1 × 10 5 cells) from 4T1 tumor explants were added (10 μg/1 × 10 5 cells) to single-cell suspensions from lymph nodes along with anti-CD3/CD28 antibodies. Proliferation of gated CD8 and CD4 T cells after 72 hours shown. G, Exosomes from human normal fibroblasts (NF) or human luminal CAFs were added (10 μg/1 × 10 5 cells) to single-cell suspensions from human PBMCs concomitantly with anti-human CD3/CD28 antibodies. Proliferation of gated CD8 and CD4 T cells shown. Experiments depict three technical replicates from at least three biological replicates. Significance was tested using one-way ANOVA with Tukey honestly significant difference post hoc test. [ A, Created in BioRender. Jones, D. (2026) https://BioRender.com/nbcfuj2 .]

Article Snippet: Next, the cells were centrifuged and plated in a 24-well plate at a concentration of 2 × 10 5 cells/well, followed by stimulation with 4 μL anti-CD3/CD28 activation beads (Miltenyi, cat. #130-093-627) for mouse.

Techniques: Labeling, Negative Control, Single Cell

Journal: Cancer Research Communications

Article Title: Collagen-Bearing Exosomes from Breast Cancer–Associated Fibroblasts Promote T-cell Dysfunction

doi: 10.1158/2767-9764.CRC-25-0259

Figure Lengend Snippet: CAF exosomes alter TCR signaling, transcriptional regulation, and metabolic profiles in CD8 + T cells. A, Differentially expressed proteins were analyzed using volcano plots for comparisons between unstimulated murine CD8 T cells and T cells stimulated with anti-CD3/CD28 antibodies, after 48 hours, with GO Biological Process (GOBP) analysis shown in B . C, Differentially expressed proteins were analyzed using volcano plots for comparisons between murine CD8 T cells stimulated with anti-CD3/CD28 antibodies for 4 hours, after 24 hours in the presence of cancer cell or CAF exosomes, with GOBP analysis shown in D . E, Heatmap showing differentially expressed phosphorylated proteins in CD8 T cells exposed to cancer cells or CAF exosomes for 24 hours, followed by stimulated with anti-CD3/CD28 antibodies for 4 hours. F, Western blot of phosphorylated (p)ZAP-70 (Y352), total ZAP-70, pSLP-76 (S376), total SLP-76, pPLC-γ (Y783), total PLC-γ in unstimulated (-) CD8 T cells, and CD8 T cells stimulated with cancer cells or CAF exosomes (Exo.) and anti-CD3/CD28 antibodies for 15 minutes or 30 minutes.

Article Snippet: Next, the cells were centrifuged and plated in a 24-well plate at a concentration of 2 × 10 5 cells/well, followed by stimulation with 4 μL anti-CD3/CD28 activation beads (Miltenyi, cat. #130-093-627) for mouse.

Techniques: Western Blot

Journal: Cancer Research Communications

Article Title: Collagen-Bearing Exosomes from Breast Cancer–Associated Fibroblasts Promote T-cell Dysfunction

doi: 10.1158/2767-9764.CRC-25-0259

Figure Lengend Snippet: Exosomal ECM from CAFs attenuates T-cell proliferation. A, Mean difference plot showing the log fold change and average abundance of each protein from CAF exosomes. Proteins with fold changes >1 are highlighted [red, up after proteinase K (PK) or blue, down after PK treatment]. Top 10 labeled. B, Gene set enrichment analysis identified proteins associated with ECM organization in CAF exosomes. C, Western blot of COL1A1 and COL5A2 proteins in cells (top) and in exosomes (bottom) of normal fibroblasts (NF) from the MFP of naïve animals and CAFs from the MCaP murine cell line. D, Western blot of COL1A1 and COL5A2 proteins in cells (top) and in exosomes (bottom) of untransformed normal human fibroblasts (NF) and CAFs from luminal breast cancer. E and F, Protein content based on BCA assay ( E ) and extracellular vesicle composition ( F ) of fractions 1–12 collected by iodixanol density gradient centrifugation. G, Western blot of fractions from ultracentrifugation, probed with antibodies against COL1A1, COL5A2, exosome markers Alix and CD63, and the serum protein albumin. H, Western blot of COL1A1 (top) and COL5A2 (bottom) in MCaP CAFs with empty vector (vector) or targeted sgRNA (sg) vectors. I, Exosomes from MCaP CAFs with empty vector or targeted sgRNA constructs were added to CFSE-labeled T cells along with anti-CD3/CD28 antibodies. CFSE dilution in T cells was measured by flow cytometry 72 hours later. J, Western blot of CAF exosomes after collagenase I digestion for 1 hour at indicated concentrations. K, CAF exosomes after digestion with collagenase I at indicated concentration were added to CFSE-labeled T cells along with anti-CD3/CD28 antibodies. CFSE dilution was measured by flow cytometry 72 hours later. Significance was tested using one-way ANOVA with Tukey honestly significant difference post hoc test ( I and K ). L, Naïve CD8 + T cells isolated from Balb/c mice were activated with anti-CD3/CD28 antibodies and nucleofected 72 hours later with Cas9 and sgRNAs targeting SHP2, DDR1, or LAIR-1, or with mock nucleofection (mock). Knockdown efficiency was assessed by Western blot. M, A subset of T cells from each cohort described in ( L ) was labeled with CFSE and subsequently stimulated with CAF-derived exosomes and anti-CD3/CD28 antibodies. Proliferation was assessed by flow cytometry 72 hours later. CFSE-labeled T cells stimulated with anti-CD3/CD28 antibodies alone served as positive controls (Stim.), whereas unstimulated cells (“Unstim.”) served as negative controls. Significance was tested using one-way ANOVA with Tukey honestly significant difference post hoc test.

Article Snippet: Next, the cells were centrifuged and plated in a 24-well plate at a concentration of 2 × 10 5 cells/well, followed by stimulation with 4 μL anti-CD3/CD28 activation beads (Miltenyi, cat. #130-093-627) for mouse.

Techniques: Labeling, Western Blot, BIA-KA, Gradient Centrifugation, Plasmid Preparation, Construct, Flow Cytometry, Concentration Assay, Isolation, Knockdown, Derivative Assay

Journal: Cancer Research Communications

Article Title: Collagen-Bearing Exosomes from Breast Cancer–Associated Fibroblasts Promote T-cell Dysfunction

doi: 10.1158/2767-9764.CRC-25-0259

Figure Lengend Snippet: ECM from CAF exosomes is critical for attenuating T-cell proliferation. A, Western blot of COL1A1 and COL1A2 in MCaP cancer cells (top) and derived exosomes (bottom) from parental cells (transduced with vector control) or cells overexpressing (OE) COL1A1 and or COL1A1/COL1A2 vectors. B, Exosomes from MCaP cancer cells expressing vector control (VC), COL1A1, and/or COL1A1/COL1A2 were added to CFSE-labeled T cells along with anti-CD3/CD28 antibodies. CFSE dilution was measured by flow cytometry 72 hours later. C and D, Exosomes from cohorts in B were added to isolated CD8 T cells along with anti-CD3/CD28 antibodies. Flow cytometry measurements of granzyme B ( C ) and IFNγ ( D ) were made after 72 hours. Significance was tested using one-way ANOVA with Tukey honestly significant difference post hoc test. E, Quantification of serum exosomes during MCaP tumor progression at 12 weeks after implantation. F, Western blot analysis of epithelial, ECM, and exosomal marker proteins from purified serum exosomes of tumor-naïve mice ( n = 4) and mice bearing MCaP tumors ( n = 5) 12 weeks after implantation. G, Single-cell suspensions from lymph nodes of naïve BALB/c mice were incubated for 72 hours with serum exosomes from tumor-naïve mice and mice bearing MCaP tumors 12 weeks after implantation. Flow cytometry was performed to measure the percentage of CD8 (left) and CD4 (right) T cells proliferating in response to concomitant exosome and anti-CD3/CD28 antibody stimulation. H, Growth curve of MCa-P1362 tumors injected weekly with PBS or 20 μg weekly of CAF exosomes. I, Quantification of CD8 (left) and CD4 (right) T cells in tumor-draining lymph nodes and primary tumors of PBS-injected and CAF exosome–injected mice. P value calculated by unpaired Student t test ( E , G , and I ) and two-way ANOVA ( H ). *, P < 0.05; ****, P < 0.0001.

Article Snippet: Next, the cells were centrifuged and plated in a 24-well plate at a concentration of 2 × 10 5 cells/well, followed by stimulation with 4 μL anti-CD3/CD28 activation beads (Miltenyi, cat. #130-093-627) for mouse.

Techniques: Western Blot, Derivative Assay, Transduction, Plasmid Preparation, Control, Expressing, Labeling, Flow Cytometry, Isolation, Marker, Purification, Single Cell, Incubation, Injection