Anti cd3 cd28 coated dynabeads

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-CD3/CD28-coated Dynabeads are magnetic beads coated with antibodies targeting the CD3 and CD28 receptors on T cells. This product is designed for the activation and expansion of T cells in vitro.

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Anti-CD3/CD28 mAb-coated Dynabeads CD3/CD28-coated Dynabeads Anti-CD3/CD28 Dynabeads Anti-CD3/anti-CD28 Dynabeads CD3/CD28 Dynabeads Anti-CD3/CD28 Dynabeads CD3 Anti-CD28 Anti-CD3 Dynabeads

12 protocols using anti cd3 cd28 coated dynabeads

Purification and Activation of Mouse T Cells

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CD3⁺ cells were purified from 2–4 months old mouse spleens using MojoSort cell isolation kit (BioLegend). T cell activation was performed using low endotoxin, azide-free (LEAF) purified anti-mouse CD3 (Clone 145–2C11; 5 μg/mL) coated to the plate and LEAF Purified anti-mouse CD28 (clone 37.51; 2 μg/mL) in complete RPMI media for 24 or 48 h. Cytokine production was measured by LEGENDplex and confirmed by ELISA.
For T cell proliferation, CD4 T cells were isolated from spleen and LN of 10-week-old mice using negative selection (StemCell Technologies, Vancouver, Canada). CellTrace CFSE-labeled T cells were cocultured with anti-CD3/CD28-coated Dynabeads (Life Technologies, Carlsbad, CA) at 1:1 ratio according to the manufacturer’s protocol in a U bottom 96-well plate for 96 h in presence of rmIL-2 (30 units/mL).

Hernández-Ruiz M., Othy S., Herrera C., Nguyen H.T., Arrevillaga-Boni G., Catalan-Dibene J., Cahalan M.D, & Zlotnik A. (2019). Cxcl17−/− mice develop exacerbated disease in a T cell-dependent autoimmune model. Journal of leukocyte biology, 105(5), 1027-1039.

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Lentiviral Transduction of Activated Human T Cells

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Purified human T cells (STEMCELL Technologies, catalog #15061) from Leuko Paks of healthy donors were obtained from the Massachusetts General Hospital (MGH) blood bank under an IRB-exempt protocol in accordance with the U.S. Common Rule. For T cell expansion, T cells were activated (day 0) with anti-CD3/CD28-coated Dynabeads (Life Technologies, Catalog #111.32D) at a 3:1 bead-to-cell ratio. 24 hours after activation, T cells were lentivirally transduced to express the CAR construct. T cells were grown in RPMI media supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin (P/S), and 20 IU/mL of recombinant human IL-2. Cultures were maintained at 0.5e⁶ cells per mL and generally passaged every other day. For functional testing, cells were cryopreserved at day 9/10 of culture and directly used after thawing.

Ormhøj M., Scarfò I., Cabral M.L., Bailey S.R., Lorrey S.J., Bouffard A.A., Castano A.P., Larson R.C., Riley L.S., Schmidts A., Choi B.D., Andersen R.S., Cédile O., Nyvold C.G., Christensen J.H., Gjerstorff M.F., Ditzel H.J., Weinstock D.M., Barington T., Frigault M.J, & Maus M.V. (2019). Chimeric antigen receptor T cells targeting CD79b show efficacy in lymphoma with or without co-targeting CD19. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(23), 7046-7057.

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Transduction of Activated T Cells

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T cells were purified from normal donors by negative selection using the RosetteSep Human CD4+ or CD8+ T Cell Enrichment Cocktails according to the manufacturer’s protocols (StemCell Technologies). T cells were cultured at 1x10⁶ per mL in “complete RPMI 1640:” RPMI 1640 (Life Technologies) supplemented (ThermoFisher Scientific) wih 10% fetal calf serum (Seradigm), 1% Penn Strep (Life Technologies), 2 mM GlutaMax (Life Technologies), and 25 mM HEPES buffer (Life Technologies). T cells were stimulated with anti-CD3/CD28 coated Dynabeads (Life Technologies) at a 3:1 bead to cell ratio and 100–300 IU/mL of recombinant human interleukin-2 for 5 days prior to bead removal. 1 day after stimulation, 200ul of lentivirus supernatant was added to 0.5x10⁶ cells so that between 40–70% of the T cells would be transduced. MMLV vector transduction was performed on days 3 and 5, with 1ml virus supernatant added to a Retronectin (Takara)-coated 24 well plate and spinoculated according to the manufacturer’s instructions. Medium was doubled on day 3 and changed completely on day 5, and then added every other day throughout cell culture, or as necessary based on cell counts.

Leibman R.S., Richardson M.W., Ellebrecht C.T., Maldini C.R., Glover J.A., Secreto A.J., Kulikovskaya I., Lacey S.F., Akkina S.R., Yi Y., Shaheen F., Wang J., Dufendach K.A., Holmes M.C., Collman R.G., Payne A.S, & Riley J.L. (2017). Supraphysiologic control over HIV-1 replication mediated by CD8 T cells expressing a re-engineered CD4-based chimeric antigen receptor. PLoS Pathogens, 13(10), e1006613.

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Proliferation Assay of PBMCs Treated with FL118

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PBMCs from healthy donors were seeded in 96-well flat-bottom plates at 4 Â 10 4 cells/well and stimulated with anti-CD3/CD28coated dynabeads (Life Technologies) in a bead-to-cell ratio of 1:3 and 50 U/mL recombinant human IL2. After 24 hours, the PBMCs were treated with serial dilutions of FL118 or a DMSO control, representing the highest DMSO content, for 24 hours. BrdUrd was added 16 hours prior to the termination of proliferation. BrdUrd incorporation was measured by Cell Proliferation ELISA (Sigma-Aldrich) according to the manufacturer's protocol.

Holthof L.C., van der Schans J.J., Katsarou A., Poels R., Gelderloos A.T., Drent E., van Hal-van Veen S.E., Li F., Zweegman S., van de Donk N.W.C.J., Themeli M., Groen R.W.J, & Mutis T. (2021). Bone Marrow Mesenchymal Stromal Cells Can Render Multiple Myeloma Cells Resistant to Cytotoxic Machinery of CAR T Cells through Inhibition of Apoptosis. Clinical cancer research : an official journal of the American Association for Cancer Research, 27(13).

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Tregs Activation Assay Protocol

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In the activation assay, Tregs were stimulated for 24 h through the TCR (positive control) with anti-CD3/CD28-coated dynabeads (1 bead:2 cells ratio) (Thermo Fisher Scientific) or through the HLA.A2-CAR with 2.5μL HLA-A2∗02 dextramer (Immudex). Cells were then harvested and analyzed by flow cytometry.

Monteferrario D., David M., Tadi S.K., Zhou Y., Marchetti I., Jeanneau C., Saviane G., Dupont C.F., Martelli A.E., Truong L.N., Eshleman J.A., Ng C.C., Huston M.W., Davis G.D., Fontenot J.D., Reik A., Rosa M.D, & Fenard D. (2024). Epigenetic control of multiple genes with a lentiviral vector encoding transcriptional repressors fused to compact zinc finger arrays. Molecular Therapy. Methods & Clinical Development, 32(2), 101255.

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T-cell Activation Modulation by ALCAM

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For our in vitro experiment, T-cell (CD4⁺CD25⁻) population was magnetically sorted to exclude the regulatory T-cell population from single-cell suspensions of splenocytes of C57BL/6 mice or human peripheral blood mononuclear cells (PBMCs; STEMCELL Technologies) using CD4⁺CD25⁺ T-cell isolation kits (Miltenyi Biotech, CA, USA). The purity of the sorted cells (>95%) was confirmed by flow cytometry analysis prior to the coculture setup. T cells cultured alone served as a negative control. For coculture assays, ^- CD4⁺CD25^- T cells, stimulated with anti-CD3/CD28-coated Dynabeads (Thermo Fisher) at 1:1 ratio, were cultured on a monolayer of MSCs in the presence or absence of human anti-ALCAM neutralizing antibody (20 µg/mL; R&D Systems, CA, USA) for 24 and 48 h to assess early T-cell activation and Th1 generation, respectively. In a separate set of experiments, cocultures were treated with human anti-CD6 or isotype controls (20 μg/m; R&D Systems) for 24 and 48 h. For murine cocultures, stimulated T cells were cultured alone or on a monolayer of Mock or ALCAM-silenced MSCs.

Cho W.J., Mittal S.K, & Chauhan S.K. (2023). Mesenchymal Stromal Cells Suppress T-Cell-Mediated Delayed-Type Hypersensitivity via ALCAM-CD6 Interaction. Stem Cells Translational Medicine, 12(4), 221-233.

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Generating CAR- or TCR-Transduced T Cells

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To generate CAR-transduced or TCR-transduced T cells, human CD8+ T cells (obtained from the Hematopoietic Cell Processing and Repository Core at the Fred Hutchinson Cancer Research Center) were stimulated with antiCD3/CD28-coated Dynabeads (ThermoFisher Scientific) for 48 h at a bead-to-cell ratio of 1:1 in the presence of 30 U/mL rIL-2. Beads were then magnetically removed and transferred to retronectin-coated plates (Takara) with virus, then spin-infected for 1 h at 3000×g at 32 °C. Following a second spinoculation in retroviral supernatant the next day, the cells were cultured for 24 h in the presence of IL-2 before using them as a tumor therapeutic.

Parayath N.N., Stephan S.B., Koehne A.L., Nelson P.S, & Stephan M.T. (2020). In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo. Nature Communications, 11, 6080.

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Gut Homing T Cell Induction

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Naïve (CD3⁺ CD4⁺ CD44^– CD62L⁺) T cells were isolated from the spleen of Drd5^+/+ or Drd5^–/– mice by cell sorting using a FACS Aria II (BD), obtaining purities over 98%. Gut tropism was imprinted by activation of T cells in the presence of RA and IL-2 as described previously.³¹ (link) Briefly, naïve T cells were resuspended (10⁶ cells/mL) in RPMI 1640 medium containing 10% FBS, 2 mM L-glutamine, 1% penicillin/streptomycin, MEM nonessential amino acids 1× and sodium pyruvate 1×, gentamicin 50 μg/mL, and β-mercaptoethanol 1 μg/mL (all from Gibco [Gaithersburg, MD], Thermo Fisher Scientific). Cells were activated with anti-CD3/CD28 coated Dynabeads (Thermo Fisher Scientific) at a beads-to-cells ratio of 1:1 in the presence of 100 nM all-trans RA (Sigma-Aldrich) and 1000 U/mL recombinant mouse IL-2 (PeproTech, Rocky Hill, NJ) for 5 days. Viability and gut tropism were routinely confirmed after 5 days of culture by staining with ZAq Fixable Viability kit (BioLegend) and CCR9 and α4β7 immunostaining followed by flow cytometry analysis.

Osorio-Barrios F., Navarro G., Campos J., Ugalde V., Prado C., Raïch I., Contreras F., López E., Espinoza A., Lladser A., Franco R, & Pacheco R. (2021). The Heteromeric Complex Formed by Dopamine Receptor D5 and CCR9 Leads the Gut Homing of CD4+ T Cells Upon Inflammation. Cellular and Molecular Gastroenterology and Hepatology, 12(2), 489-506.

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T-cell Proliferation Assay on MSCs

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To assess proliferation, sorted T cells, labeled with CellTrace CFSE proliferation kit (Thermo Fisher Scientific, MA, USA), were stimulated with anti-CD3/CD28-coated Dynabeads (Thermo Fisher) at 1:1 ratio and cultured on a monolayer of MSCs, as described above, for 72 h. T cells, harvested and surface stained for CD4 surface marker, were acquired using an LSR II flow cytometer (BD Biosciences). The proliferation index was calculated as total number of divisions divided by the number of cells that underwent division using FlowJo software (BD Biosciences).

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Adoptive Transfer of Tumor-Specific T Cells

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Adoptive transfers were performed as described previously [25 (link)]. The number of transferred cells and the timing of transfer and infection are stated in each figure legend. For the experiments in Fig. 6 and Suppl. Fig. 3A, OT-Is and Pmel-Is were co-transferred into mice one day before implantation of B16F0s. Recipients were co-infected with MCMV-SL8 and MCMV-gp100 5 days after tumor implantation to expand both OT-Is and Pmel-Is. For in vitro activation, OT-Is were either taken from fresh or frozen naïve splenocytes (frozen in T cell media plus 10% DMSO) and enriched via magnetic negative CD8 selection as described above. Enriched cells were activated with anti-CD3/CD28 coated Dynabeads (ThermoFisher Scientific) following the manufacturer's protocol in T cell media containing 30 U/ml rIL-2 (Biolegend). Upon transfer, OT-Is were 90–100% alive (determined by Zombie Aqua™ Fixable Viability Kit [Biolegend]), 95–100% CD44^hi (determined by anti-CD44 [clone IM7]), and had expanded 20–30 times.

Erkes D.A., Smith C.J., Wilski N.A., Caldeira-Dantas S., Mohgbeli T, & Snyder C.M. (2017). Virus-specific CD8+ T cells infiltrate melanoma lesions and retain function independent of PD-1 expression. Journal of immunology (Baltimore, Md. : 1950), 198(7), 2979-2988.

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