Primary human CD3+ T cells were negatively selected from single donor PBMCs using standard techniques. For revival and culture, 5.0 × 106 cryopreserved T cells were expanded using CD3/CD28 T cell activator (StemCell Technologies) and standard conditions for 16 days to achieve sufficient cell numbers for the experimental workflow. Further details outlined in SI.
Cd3 cd28 t cell activator
Manufactured by STEMCELL
Sourced in Canada, United States
The CD3/CD28 T cell activator is a laboratory product designed to stimulate and activate T cells. It consists of antibodies that bind to specific cell surface proteins, CD3 and CD28, which are important for T cell activation and proliferation. This product provides a controlled and standardized method for T cell activation in research and experimental settings.
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Lab products found in correlation
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Ficoll paque plus gradient, by GE Healthcare (1 mentions)
Exosome depleted fbs, by Thermo Fisher Scientific (1 mentions)
Automacs, by Miltenyi Biotec (1 mentions)
Rpmi medium, by Lonza (1 mentions)
Annexin 5 fitc apoptosis staining detection kit, by Abcam (1 mentions)
Cd8 t cell isolation kit, by STEMCELL (1 mentions)
Interleukin 2 (il 2), by R&D Systems (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Cd40l, by Cell Signaling Technology (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Human naive cd4 t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Rhil 2, by STEMCELL (1 mentions)
Ficoll paque plus, by Merck Group (1 mentions)
Cytoflex flow cytometry, by Beckman Coulter (1 mentions)
Fixable viability kit, by BioLegend (1 mentions)
Annexin 5 assay, by Beckman Coulter (1 mentions)
12 protocols using cd3 cd28 t cell activator
1
Expansion of Primary Human T Cells
2
Isolation and Activation of Human T Cells
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Peripheral blood was obtained from healthy volunteers who signed a consent form approved by the Institutional Review Board (IRB no. 0403105) at the University of Pittsburgh. Venous blood was collected in heparin tubes and was processed immediately for recovery of peripheral blood mononuclear cells (PBMC) by centrifugation on Ficoll-Paque Plus gradients (GE Healthcare Bioscience, Pittsburgh, PA, USA). The recovered cells were washed in RPMI medium (Lonza, Basel, CH) supplemented with 10% (v/v) exosome-depleted FBS (Gibco, Thermo Scientific, Pittsburgh, PA, USA) and immediately used for experiments. T cells were isolated by negative selection on AutoMACS (Miltenyi Biotec, San Diego, CA, USA) using an isolation kit from Miltenyi Biotec as previously described by us [52 (link)]. T cells were activated using CD3/CD28 T cell activator (25 μL/mL, Stemcell, Vancouver, BC, CA) and IL-2 (150 IU/mL, PeproTech, Bionity, Rocky Hill, CT, USA) in freshly-prepared RPMI for 48–72 h prior to culture for exosome production.
3
Exosome-induced CD8+ T cell apoptosis in HNSCC
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CD8+ T cells (2 × 106/mL), isolated with CD8+ T cell isolation kit (17953, Stemcell Technologies, Vancouver, Canada) according to manufacturer’s instructions, were activated using CD3/CD28 T-cell activator (25 μL/mL, Stemcell Technologies) and 0.7 µL IL2 (2 × 105 U/ml, R&D Systems, Minneapolis, MN, USA) in RPMI supplemented with exosome-depleted FBS (Gibco, Waltham, MA, USA) for 6 or 16h, respectively, for activation or apoptosis assay. Next, exosomes (50 μL of the total fraction, CD45(+), or CD45(−) fraction) isolated from plasma of HNSCC patients were added and co-cultures were incubated for 16 or 6h, respectively, for activation or apoptosis assay. Only the CD45(+) fraction was bound on beads. Co-cultures containing no exosomes (50 μL PBS alone) or streptavidin beads with biotinylated CD45Ab served as negative controls. Apoptosis of CD8+ T cells and CD69 expression were measured by flow cytometry with a Gallios flow cytometer using an apoptosis assay kit (Annexin V-FITC Apoptosis Staining/Detection Kit, Abcam, Cambridge, UK) and CD69-FITC antibody (555530, BD Pharmingen, Franklin Lakes, NJ, USA), respectively. The protocol was previously described in detail [14 (link)].
4
Isolation and Activation of T Cell Subsets
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Treg, CD4+ T cells and CD8+ T cells were isolated from PBMC. CD4+ T and CD8+ T cells were activated by CD3/CD28 T Cell Activator (StemCell) for 48 h.
In vitro induced Treg (iTreg) were induced from CD4+ T cells. CD4+ T cells were activated by CD3/CD28 T Cell Activator for 7 days in X-Vivo 15 medium containing 10% FBS, 1% Glutamax, 2 mg/mL N-acetylcysteine, 1 × sodium pyruvate, 1 × HEPES, 1 × nonessential amino acids, 1 × pen/strep, 50 μM 2-mercaptoethanol, 500 U/mL IL2, 100 ng/mL rapamycin and 10 ng/mL TGFβ1. For CD25 expression assay, CD4+, CD8+ naïve T cells and activated CD4+T, CD8+T cells were stained by anti-human CD4 or CD8 and CD25 fluorescent-labeled antibody. Treg and iTreg were stained by anti-human CD3, CD4, Foxp3 and CD25 fluorescent-labeled antibody. Cells were analyzed by Beckman CytoFLEX flow cytometry.
Methods of ADCC assay for these cell types was the same as described in “In vitro ADCC assay ”.
In vitro induced Treg (iTreg) were induced from CD4+ T cells. CD4+ T cells were activated by CD3/CD28 T Cell Activator for 7 days in X-Vivo 15 medium containing 10% FBS, 1% Glutamax, 2 mg/mL N-acetylcysteine, 1 × sodium pyruvate, 1 × HEPES, 1 × nonessential amino acids, 1 × pen/strep, 50 μM 2-mercaptoethanol, 500 U/mL IL2, 100 ng/mL rapamycin and 10 ng/mL TGFβ1. For CD25 expression assay, CD4+, CD8+ naïve T cells and activated CD4+T, CD8+T cells were stained by anti-human CD4 or CD8 and CD25 fluorescent-labeled antibody. Treg and iTreg were stained by anti-human CD3, CD4, Foxp3 and CD25 fluorescent-labeled antibody. Cells were analyzed by Beckman CytoFLEX flow cytometry.
Methods of ADCC assay for these cell types was the same as described in “
5
Isolation and Activation of Immune Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of HDs by centrifugation on Ficoll-Paque Plus (GE Healthcare Lifesciences) gradients38 (link). CD4+ T, CD8+ T, NK and B cells were isolated from PBMCs using negative selection-based cell isolation kits from Miltenyi, #130-096-495, #136-096-533, Stem Cell Technologies, #19055, and Biolegend #480061, respectively, following manufacturers’ protocol. Isolated cells were cultured in RPMI medium supplemented with 10% (v/v) exosome-depleted and heat-inactivated FBS at 37 °C in the atmosphere of 5% CO2 in air16 (link). T cells were activated with CD3/CD28 T cell activator (25 µl/mL, Stem Cell) and IL-2 (150 IU/mL, Peprotech) at 1 × 106 cells/mL in RPMI medium for 12 h. NK cells were activated by 48 h incubation with IL-2 (100 IU/mL, Peprotech) and IL-15 (150 IU/mL, Peprotech) at 1 × 106 cells/mL. B cells were activated by 12 h incubation with IL-4 (100 IU/mL, R & D System) and CD40L (0.5 ug/mL, Cell Signaling Technology, #3583 S) at 1 × 106 cells/mL.
6
Isolation and Activation of Human Naive T Cells
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Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Paque Plus (Sigma Aldrich). Conventional T cells (CD4+CD25−CD45RA+CD45RO−) were isolated using human naive CD4+ T cell isolation kit II (Miltenyi Biotec). Purity was >97% as confirmed by flow cytometry. Purified Tconv cells were cultured in RPMI 1640 medium with rhIL-2 and CD3/CD28 T Cell Activator (Stemcell Technologies).
7
Activation and Analysis of Human T Cells
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Human CD3+ T cells were activated by CD3/CD28 T Cell Activator (StemCell) for 48 h in the presence of 10 μg/mL CD25 antibody in 1640 + 10% FBS (Gibco) medium. After staining by Fixable Viability Kit (Biolegend, 423105) for 15 min, cells were washed by two times. Cells were then stained by mixture of anti-human CD3, CD4 and CD8 fluorescent-labeled antibody at 4 °C for 20 min and washed. After resuspending by 1 × Fixation/Permeabilization buffer, cells were incubated in dark for 30 min at room temperature and then resuspended by 1 × Permeabilization buffer. After centrifugation, cells were stained by Ki67 and Granzyme B fluorescent-labeled antibody for 30 min and then analyzed by Beckman CytoFLEX flow cytometry. Experiments were performed in triplicate, value = mean ± SEM.
8
CD8+ T cell Apoptosis Assay
9
Human PBMC-derived T Cell Activation
10
T-Cell Proliferation Assay with Macrophage Conditioned Media
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T cells were isolated from the spleens of female C57BL/6 mice using a Pan T-Cell Isolation Kit (Miltenyi Biotec, CA, USA). Then, carboxyfluorescein succinimidyl ester (CFSE) (Sigma‒Aldrich, MO, USA)-labeled T cells were cultured with 10 ng/ml IL-2 (PeproTech, NJ, USA) in complete RPMI-1640 medium (10% FBS, 100 U/ml penicillin–streptomycin) and stimulated with CD3/CD28 T-Cell Activator (Stemcell Technologies, BC, Canada) in the presence or absence of conditioned medium from control or APG-2575-treated BMDM-M2 cells. After 72 h, CFSE was detected, and T cells were labeled with CD8 for specific measurement of T-cell proliferation. In some instances, macrophage-induced tumor cell apoptosis or APG-2575-induced macrophage apoptosis was measured by Annexin-V and PI staining (BD Pharmingen).
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