Lentivirus vector construction and lentivirus production were established as previously described (Hu et al., 2021 (link)). To generate TCR-transduced-CD4+ T and TCR-transduced-CD4+ Jurkat cells, CD4+ T cells were stimulated with 1 μg/ml OKT3 antibody and 1 μg/ml anti-CD28 antibody (Miltenyi Biotec) and cultured in a complete medium containing 100 IU/ml IL-2 for 48 h. CD4+ Jurkat cells and stimulated CD4+ T cells were seeded into 24-well-plates and incubated with 500 μl lentivirus and 500 μl RPMI 1640 for 6 h. Next, 500 μl complete medium was added to each well. After 24 h, the original medium was replaced with a 2-ml complete medium and incubated for 48 h. Then, the cells were harvested and used for subsequent assays.
Anti cd28 antibody
Manufactured by Miltenyi Biotec
Sourced in Germany
The Anti-CD28 antibody is a laboratory reagent used for the activation and stimulation of T cells. It targets the CD28 receptor, a co-stimulatory molecule expressed on the surface of T cells, to enhance their proliferation and cytokine production.
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Lab products found in correlation
Anti cd3 antibody, by Miltenyi Biotec (3 mentions)
Anti cd3, by Miltenyi Biotec (3 mentions)
Apc conjugated anti human cd3, by BioLegend (2 mentions)
Hil 2, by Miltenyi Biotec (2 mentions)
Anti cd3, by Thermo Fisher Scientific (2 mentions)
Okt3 antibody, by Miltenyi Biotec (1 mentions)
Aim 5, by Thermo Fisher Scientific (1 mentions)
Anti il 4 antibody, by R&D Systems (1 mentions)
Cd4 t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Na ve cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Interleukin 2 (il 2), by R&D Systems (1 mentions)
Il 12, by R&D Systems (1 mentions)
Human cd4 microbeads, by Miltenyi Biotec (1 mentions)
Anti cd28 antibody, by Thermo Fisher Scientific (1 mentions)
Facs canton 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Anti il 10, by R&D Systems (1 mentions)
Proleukin, by Novartis (1 mentions)
Facsaria system, by BD (1 mentions)
9 protocols using anti cd28 antibody
1
Generation of TCR-transduced CD4+ T Cells
2
Isolation of PRAME-specific CD137+ T cells
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50 × 106 viable PBMC or G‐MNC was resuspended in 1 mL of AIM‐V (Gibco‐BRL, CA, USA) supplemented with 10% human AB serum (Australian Red Cross Lifeblood, Sydney, Australia). PRAME peptide mixture was added to PBMC to a concentration of 1 µg mL–1 per peptide, and anti‐CD28 antibody (Miltenyi Biotec) was added to a concentration of 1 µg mL–1. The cells were incubated at 37°C at 5% CO2 for 1 h. The cultures were then incubated in a 6‐well plate for a further 16–24 h at concentration of 1 × 107 mL–1 by adding medium with CD28 antibody to maintain concentration of 1 µg mL–1. CD137+ cells were labelled with CD137‐PE and anti‐PE MicroBeads (Miltenyi Biotec) as per manufacturer’s instructions. CD137+ cells were isolated by positive selection with MS MACS columns using miniMACS magnetic separators.
3
Isolation and Differentiation of Naïve and Memory CD4+ T Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque density gradient centrifugation. CD4+ T cells were isolated by depletion of non-CD4+ T cells using a CD4+ T Cell Isolation Kit II (Miltenyi Biotec). The cells were then separated into naïve and memory CD4+ T cells using a Naïve CD4+ T cell Isolation Kit (Miltenyi Biotec). Purification of the cells was confirmed by labeling samples before and after purification with fluorescently labeled antibodies to either CD4 and CD45RA (to label naïve cells) or CD4 and CD45RO (to label memory cells) and analysis using flow cytometry. 94.3% of the cells in the naïve T cell preparations were CD4+ (SE = 0.7%, ranging from 83.9% to 98.6%) and 83.8% were CD45RA+ (SE = 1.4%, ranging from 68.1% to 95.9%). 95.2% of the cells in the memory T cell preparations were CD4+ (SE = 0.4%, ranging from 89.7% to 98%) and 75.0% were CD45RO+ (SE = 1.8%, ranging from 55.0% to 88.6%). Cells were plated in 24-well dishes coated with 2.5 µg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% FCS, 2.5 µg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems). For TH1 differentiation, the media also included 20 ng/ml IL-12 and 1 µg/ml anti-IL-4 antibody (R&D Systems). For TH2 differentiation, the media also included 20 ng/ml IL-4 and 2 µg/ml anti-IL-12 antibody (R&D Systems). Cells were harvested after three days.
4
Isolation and Activation of Human T Cells
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Whole blood was taken from healthy donors. Using Ficoll–Paque density gradient centrifugation, human peripheral blood mononuclear cells (PBMCs) were isolated. Isolated PBMCs were seeded into 24-well plates (1.5 × 106 cells/well) and cultured in RPMI1640 containing 10%FBS and 100 IU hIL-2 (Miltenyi Biotec). To activate and enrich T cells, PBMCs were cultured with 3 ug/ml anti-CD3 antibody (Miltenyi Biotec) and 10 ug/ml anti-CD28 antibody (Miltenyi Biotec). After 4 days of incubation at 37 °C, the purity of T cells was assayed using APC conjugated anti-human CD3 (BioLegend, USA) by flow cytometry. The clone of APC anti-human CD3 Antibody was UCHT1.The purity of T cells is represented in the figure S 2 .
5
Isolation and Activation of Human T Cells
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Whole blood was taken from healthy donors. Using Ficoll-Paque density gradient centrifugation, human peripheral blood mononuclear cells (PBMCs) were isolated. Isolated PBMCs were seeded into 24-well plates (1.5×10 6 cells/well) and cultured in RPMI1640 containing 10%FBS and 100IU hIL-2 (Miltenyi Biotec). To activate and enrich T cells, PBMCs were cultured with 3 ug/ml anti-CD3 antibody (Miltenyi Biotec) and 10 ug/ml anti-CD28 antibody (Miltenyi Biotec). After 4 days of incubation at 37°C purity of T cells was assayed using APC conjugated anti-human CD3 (BioLegend, USA).
6
Isolation and Activation of CD4+ T Cells
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PBMCs were isolated from heparinized blood samples by Ficoll–Hypaque density-gradient centrifugation. Peripheral CD4+ T cells of controls were isolated from PBMCs by human CD4 microbeads (Miltenyi Biotec, Palo Alto, CA, USA) according to the manufacturer’s instructions. The purity of CD4+ T cells was detected using FACS Canton II and FACS Diva software (BD Company, Franklin Lakes, NJ, USA) after staining with anti-human CD4 antibodies (BD Company, USA). The result showed that the purity was >92% in each experiment. PBMCs were cultured for 72 h with or without rhIFN-α 2a (PBL Biomedical Lab, Piscataway, NJ, USA), anti-IL-10 (R&D Systems) at a concentration of 1 × 106 cells/ml in combination with anti-CD3 (5 mg/ml) and anti-CD28 anti- bodies (1 mg/ml) (eBioscience, San Diego, CA, USA). CD4+ T cells (1 × 106 cells/ml) were cultured for 72 h with or without rhIFN-α 2a (1 mg/ml) in combination with anti-CD3 (5 mg/ml) and anti-CD28 antibodies (1 mg/ml) (Miltenyi Biotec,Germany). Supernatants from these cell cultures were used for detecting the concentration of IL-17 and IL-10.
7
T Cell Activation and Transduction
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PBMCs were suspended in RPMI containing FCS and L-glutamine at a concentration of 1 × 106 cells/mL. They were activated with 0.5 μg/mL of anti-CD3 (Miltenyi Biotec) and anti-CD28 antibodies (Miltenyi Biotec). Forty-eight hours before transduction and on the day of transduction, 100 IU/mL recombinant human IL-2 (Proleukin, Novartis) was added. T cells were transduced using γ-retroviral transduction. Transduction efficiency was measured 3 days after transduction using flow cytometry by staining for CD34 (R&D). T cell populations were not corrected for transduction efficiency in functional assays.
8
PBMC Isolation and Cytokine Production
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Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using lymphocyte-separation medium (ICN/Cappel Pharmaceuticals, Bridgewater Township, NJ, USA). PBMCs were resuspended in phosphate-buffered saline (PBS)/3% human IgG (Baxter International, Deerfield, IL, USA) to block Fc receptors and prevent non-specific antibody binding, and incubated for 30 min at 4°C in the dark. Cells were then washed with PBS containing 1% bovine serum albumin (BSA). Background fluorescence was assessed using appropriate isotype- and fluorochrome-matched control monoclonal antibodies (BD Biosciences, San Jose, CA, USA). Cytokine production was assessed after stimulation of cells (1 × 105 cells/200 μL) for 48 h with anti-CD3 (2 μg/mL; eBiosciences) and anti-CD28 antibodies (0.5 μg/mL; Miltenyi Biotec, Bergisch Gladbach, Germany).
Antibodies are shown in Supplementary Table2 . Cells were sorted using the FACSAria system (BD Biosciences) and analyzed using FACSVerse (BD Biosciences) and FlowJo (Tree Star, Ashland, OR, USA).
Antibodies are shown in Supplementary Table
9
Exosome Uptake by CD4+ T Cells in Buruli Ulcer Patients
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A PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich) was utilized to label CD4+T cells, and a PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich) was utilized to label exosomes. CD4+T cells (5 × 105/well) were seeded in 12-well plate(4.5 cm2/well), cultured in 500 µL exosome-free medium (UmiBio, Shanghai, China), then activated with 1 µg/mL anti-CD3 and 1 µg/mL anti-CD28 antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany). Finally, they were treated with 20 µL exosomes (1 × 1010 particles/20 µL/well) for 24 hours. The uptake of labeled exosomes was detected by an inverted fluorescence microscope (LAS X3.4.7; Leica Camera, Wetzlar, Germany) and by 520-nm and 580-nm lasers. To compare the uptake of exosomes by CD4+T cells between patients with BU and the healthy controls, a PKH26 Red Fluorescent Cell Linker Kit was utilized to label exosomes and anti-human CD4-APC antibody (clone RPA-T4; BioLegend, San Diego, CA, USA) was used to stain CD4+T cells. The relative quantification of exosome uptake by CD4+T cells was evaluated by FCM analysis.
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