Anti cd3

Manufactured by Miltenyi Biotec

Sourced in Germany, United States

Anti-CD3 is a monoclonal antibody that binds to the CD3 complex on the surface of T cells. It is used as a research tool to activate and study T cell function.

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Anti-CD3 antibody (16 citations) Anti-CD3 antibody (OKT-3, (4 citations) Anti-CD3/CD28 antibodies (4 citations) Anti-CD3/28 beads (4 citations) Anti-CD3 and anti-CD28 soluble antibody (3 citations) TAE) beads (anti-CD2/CD3/CD28) (2 citations) Anti-CD3–PerCP-Vio700 (2 citations) Anti-CD3 antibody (750 ng/24-well culture plate, (2 citations) Anti-CD3 paramagnetic beads (2 citations) Anti-CD2/anti-CD3/anti-CD28 antibody conjugated beads (1 citations)

63 protocols using anti cd3

Library Generation of Engineered TCRs

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After bulk TCR sequencing, top rank alpha chains and beta chains were selected and randomly paired to obtain a library of 116 candidate TCR constructs. Detailed information on the library can be found in Table S3. The TCR library was cloned into lentiviral vectors for T‐cell transduction. Multiplicity of infection (MOI) was controlled at 0.2 to ensure single lentivirus integration into the genome of each transduced T cell.
Lentiviruses were generated using the pMD2.G and pSPAX2 envelope plasmids in HEK293T cells. Freshly isolated PBMCs were activated with anti‐CD3 and anti‐CD28 (Miltenyi Biotec) overnight, and proceeded to virus infection. Viral supernatant mixed with 400 U/ml IL‐2 (R&D System, USA) and 8 μg/ml protamine (Sigma Aldrich) was added onto peripheral blood lymphocytes (PBLs) and centrifuged at 800 g and 25°C for 1.5 h. Transduced T cells were maintained in GT551‐H3 medium (TaKaRa) supplied with 5% human serum (GemCell), 1% penicillin/streptomycin (HyClone), and 400 IU/ml IL‐2. The expression of TCR genes was routinely examined using a mouse TCRβ‐specific antibody (BD Biosciences) by FACS.

Jiang J., Xia M., Zhang L., Chen X., Zhao Y., Zeng C., Yang H., Liang P., Li G., Li N., Qi H., Wei T, & Ren L. (2022). Rapid generation of genetically engineered T cells for the treatment of virus‐related cancers. Cancer Science, 113(11), 3686-3697.

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Multiparametric Flow Cytometry Analysis

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Primary antibodies: anti-MET (Human HGFR/c-MET APC-conjugated Antibody, clone 95,106, R&D System); anti-IgG1/CH2CH3 regions (Alexa Fluor® 647 AffiniPure F (ab’)2 Fragment Goat Anti-Human IgG (H + L) antibody, Jackson Immuno Research); anti-CD4 (APC Mouse Anti-Human CD4 clone M-T466, Miltenyi); anti-CD3 (PE Mouse Anti-Human CD3, Clone HIT3a); anti-CD8 (APC Mouse Anti-Human CD8, Clone RPA-T8); anti-CD56 (PE Mouse Anti-Human CD56, Clone MY31) all from BD Biosciences. Isotype control antibodies: APC, FITC, or PE mouse IgG1 κ Isotype Control, Clone MOPC-21 (BD Biosciences). Cells were counterstained with DAPI and analyzed by Cyan ADP flow cytometer (Beckman Coulter S.r.l.). Data were elaborated using Summit 4.3 software (Dako). For plots in which the isotype control is not shown, the Mean Fluorescence Intensity (MFI) derived from the Isotype control was set within the first logarithm (0 < MFI < 10). Cells were considered positive for the analyzed marker if the signal was higher than 10 (MFI > 10).

Chiriaco C., Donini C., Cortese M., Ughetto S., Modica C., Martinelli I., Proment A., Vitali L., Fontani L., Casucci M., Comoglio P.M., Giordano S., Sangiolo D., Leuci V, & Vigna E. (2022). Efficacy of CAR-T immunotherapy in MET overexpressing tumors not eligible for anti-MET targeted therapy. Journal of Experimental & Clinical Cancer Research : CR, 41, 309.

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Expanding and Transducing Natural Killer Cells from Umbilical Cord Blood

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UCB units were provided from StemCyte under IRB-approved protocols. All donors provided written informed consent, which followed the ethical guidelines of the Declaration of Helsinki. NK cells were isolated by using the RosetteSep^TM human NK cell enrichment cocktail (Cat# 15065, StemCell Technologies) and Ficoll-Paque (Cat# 17144003, Cytiva). The purity of primary NK cells was confirmed with flow cytometry using anti-CD56 (Cat# IM2474U, Beckman Coulter; 1:20 dilution) and anti-CD3 (Cat# 130-113-134, Miltenyi Biotec; 1:50 dilution) antibodies. Frozen UCB NK cells were thawed and expanded with irradiated K562 feeder cells expressing membrane-bound IL-21 and 4-1BBL (APC K562) in the presence of recombinant human IL-2 (50 IU/ml; NIH) in Stem Cell Growth Medium (SCGM) (Cat# 20802-0500, CellGenix). Expanded NK cells were transduced with retrovirus on day 5 in RetroNectin (Cat# T202, Takara Bio)-coated plates, according to the manufacturer’s protocol. On day 8, NK cells were co-cultured with irradiated APC K562 cells for an additional 7 days prior to being harvested for in vitro analysis or frozen (liquid nitrogen) for in vitro and in vivo studies. All NK cells used in our study did not undergo further purification, except where specifically indicated. Information on flow antibodies was presented in Supplementary Table 1.

Lu T., Ma R., Dong W., Teng K.Y., Kollath D.S., Li Z., Yi J., Bustillos C., Ma S., Tian L., Mansour A.G., Li Z., Settles E.W., Zhang J., Keim P.S., Barker B.M., Caligiuri M.A, & Yu J. (2022). Off-the-shelf CAR natural killer cells secreting IL-15 target spike in treating COVID-19. Nature Communications, 13, 2576.

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Retroviral Transduction of Activated T Cells

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Buffy coats from healthy donors were obtained through the Gulf Coast Regional Blood Center, Houston, TX, USA. Peripheral blood mononuclear cells (PBMCs) isolated with Lymphoprep density separation (Fresenius Kabi Norge) were activated using 1 μg/mL anti-CD3 (Miltenyi Biotec) and 1 μg/mL anti-CD28 (BD Biosciences) mAb-coated plates. On day 3, T lymphocytes were transduced with retroviral supernatants using retronectin-coated plates (Takara Bio, Shiga, Japan). After removal from retronectin plates, T cells were expanded in complete medium (45% RPMI 1640 and 45% Click’s medium [Irvine Scientific], 10% FBS [Hyclone], 2 mM GlutaMAX, 100 U/mL penicillin, and 100 μg/mL streptomycin) with IL-7 (10 ng/mL; PeproTech) and IL-15 (5 ng/mL; PeproTech), changing medium every 2–3 days. On days 12–14, cells were collected for in vitro co-culture experiments.

Krokhotin A., Du H., Hirabayashi K., Popov K., Kurokawa T., Wan X., Ferrone S., Dotti G, & Dokholyan N.V. (2019). Computationally Guided Design of Single-Chain Variable Fragment Improves Specificity of Chimeric Antigen Receptors. Molecular Therapy Oncolytics, 15, 30-37.

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VEGF-A Modulation of CD8+ T Cell Activity

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CD8⁺ T lymphocytes were purified from splenocytes using a CD8⁺ isolation kit (Miltenyi Biotec). Purified CD8⁺ T lymphocytes were cultured in the presence of plate-bound anti-CD3 (10 µg/ml) with or without recombinant murine VEGF-A (50 ng/ml; Miltenyi Biotec). After 48 h of culture, cells were harvested and analyzed by cytometry or used to extract mRNA. In some experiments, anti–VEGF-R1 (20 µg/ml; R&D Systems) or anti–VEGF-R2 (10 µg/ml; clone 91202; R&D Systems) antibodies or isotype control were added to the culture medium. In some experiments, 11R-VIVIT (Merck Millipore) was added 1 h at 5 µM before the addition of VEGF-A and during the stimulation with VEGF-A.

Voron T., Colussi O., Marcheteau E., Pernot S., Nizard M., Pointet A.L., Latreche S., Bergaya S., Benhamouda N., Tanchot C., Stockmann C., Combe P., Berger A., Zinzindohoue F., Yagita H., Tartour E., Taieb J, & Terme M. (2015). VEGF-A modulates expression of inhibitory checkpoints on CD8+ T cells in tumors. The Journal of Experimental Medicine, 212(2), 139-148.

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Comprehensive C2C12 Myoblast Profiling

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Cell cycle/Edu experiments were performed by incubating growth phase C2C12 myoblast cultures with 10uM 5-ethynyl-2′-deoxyuridine (EdU) for 2 hours, fixing, counterstaining DNA with DAPI, and identifying Edu+ cells using a Click-IT Edu Flow Cytometry Kit (Thermo Fisher). Mitochondria were labeled in live cells using MitoTracker Green FM (Thermo Fisher) and analyzed by flow cytometry. Immune cell profiling was performed as follows: hindlimb muscles were enzymatically digested and filtered according to standard protocols (Bernet et al, 2014 ), cells were labeled with fluorescently conjugated primary antibodies according to manufacturer’s instructions, and flow cytometry performed on a MACSquant 10 analyzer. The following antibodies (Miltenyi Biotec) were used in this study: anti-CD45, anti-CD3, anti-CD49b, anti-CD11c, anti-MHC class II, anti-F4/80, anti-CD11c, and anti-GR1. The following is the gating strategy used to identify individual immune cell subtypes: NK: CD45+, CD3e−, CD49b+; Dendritic cell: CD45+, CD11c+, MHC class II+; M1 macrophage: CD45+, F4/80+, CD11b+, CD11c+; M2 macrophage: CD45+, F4/80+, CD11b+, CD11c−; Neutrophil: CD45+, CD11b+, GR1+; Pan T cells: CD45+, CD3e+; Cytotoxic T cells: CD45+, CD3e+, CD8+; Helper T cells: CD45+, CD3e+, CD4+; Regulatory T cells: CD45+, CD3e+, CD4+, CD25+. Samples were analyzed using either MACSquant software or FlowJo v10.

Hogan K.A., Cho D.S., Arneson P.C., Samani A., Palines P., Yang Y, & Doles J.D. (2017). Tumor-derived cytokines impair myogenesis and alter the skeletal muscle immune microenvironment. Cytokine, 107, 9-17.

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Isolation and Purification of PBMC Subsets

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Human PBMCs were isolated using Ficoll-Hypaque® density gradient centrifugation of heparinized venous blood from donors. The PBMCs were subjected to sequential isolation of T cells, NK cells and monocytes using anti-CD3, anti-CD56 and anti-CD14 microbeads according to manufacturer’s guidelines (Miltenyi Biotec Inc., San Diego, CA). Then the rest of the cells were further separated using a human B cell isolation kit II purchased from Miltenyi Biotec Inc. to obtain B cells and dendritic cells. In some experiments, we used antigen presenting cells (APCs, i.e. a mix of B cells, dendritic cells and monocytes) isolated from PBMCs by depletion of T cells and NK cells.

Bin L., Li X., Richers B., Streib J.E., Hu J.W., Taylor P, & Leung D.Y. (2018). ANKRD1 regulates innate immune responses against herpes simplex virus 1: A potential role in eczema herpeticum. The Journal of allergy and clinical immunology, 141(6), 2085-2093.e1.

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Treg-mediated Suppression of Tresponsive Cells

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CD4⁺ T cells were enriched using the EasySep Human CD4⁺ T cell enrichment kit (STEMCELL Technologies). CD4⁺CD25^hiCD127^lo Tregs and CD4⁺CD25⁻CD127⁺ responder T cells (Tresp) were sorted by fluorescence activated cell sorting (FACS) and then labeled with 5 μM of CFSE using the CellTrace CFSE Cell Proliferation kit (Invitrogen). Treg and Tresp cells were co-cultured at a 1:1 ratio in the presence of beads loaded with anti-CD2, anti-CD3, and anti-CD28 (Human Treg Suppression Inspector; Miltenyi Biotec). On day 4.5, proliferation of the Tresp cells was analyzed by flow cytometry measuring CFSE dilution.

Minto H., Mensah K.A., Reynolds P.R., Meffre E., Rubtsova K, & Gelfand E.W. (2019). A Novel ATM Mutation Associated with Elevated Atypical Lymphocyte Populations, Hyper-IgM, and Cutaneous Granulomas. Clinical immunology (Orlando, Fla.), 200, 55-63.

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Purification of Eosinophils from Blood

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Eosinophils were purified by negative selection as previously described [39 (link)]. Briefly, heparinized blood was diluted 1 : 1 in HBSS and was overlaid above Percoll (1.090 g/ml). After centrifugation at 700 × g for 20 min at room temperature, the mononuclear cells were removed from the plasma/Percoll interface and erythrocytes were eliminated from the cell pellet by hypotonic lysis with water. The remaining pellet was resuspended in 2% NCS in HBSS. Cells were then incubated with anti-CD16, anti-CD3, anti-CD14, and anti-Glycophorin-A beads from Miltenyi Biotec (San Diego, CA) and run through an AutoMACS (Miltenyi Biotec). Eosinophil preparations with purity > 99% and viability~98% were cultured the same day, ~6-8 h after the blood draw.

Esnault S., Kelly E.A., Johnson S.H., DeLain L.P., Haedt M.J., Noll A.L., Sandbo N, & Jarjour N.N. (2019). Matrix Metalloproteinase-9-Dependent Release of IL-1β by Human Eosinophils. Mediators of Inflammation, 2019, 7479107.

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FACS Immunophenotyping of Immune Cells

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For FACS surface staining, cells were washed in staining buffer (SB) (2% FBS; 0,1% sodium azide in PBS) and after a blocking of 10 min with SB + Ab serum 20%, were stained for 30 min with mouse monoclonal antibodies. The antibodies used were: anti CD3, CD4, CD8, CD14, CD45, CD11C, EPCAM, PD-1, PD-L1, MHC-I, CD-107A (Miltenyi Biotec). Stained cells were washed 2 times, resuspended in SB and then acquired on a FACS ACCURI C6 (BD Biosciences). Analysis was conduced using accuri c6 software (BD Biosciences). The analysis of intracellular cytokine production was done after 6 h of stimulation with with phorbol 12-myristate 13-acetate (PMA, 10 ng/mL), Ionomycin (500 ng/mL) and Brefeldin A (BFA 10 μg/mL) (Sigma Aldrich) and the intracellular staining was performed incubating T cells with mouse monoclonal antibody IFNg (Miltenyi Biotech).

Della Corte C.M., Barra G., Ciaramella V., Di Liello R., Vicidomini G., Zappavigna S., Luce A., Abate M., Fiorelli A., Caraglia M., Santini M., Martinelli E., Troiani T., Ciardiello F, & Morgillo F. (2019). Antitumor activity of dual blockade of PD-L1 and MEK in NSCLC patients derived three-dimensional spheroid cultures. Journal of Experimental & Clinical Cancer Research : CR, 38, 253.

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