Dynabeads cd3 cd28 t cell expander

Naive CD4⁺ T cells were cultured in 96-well round-bottomed plates (Corning, New York City, NY, USA) at a density of 5 × 10⁴ per well in X-VIVO 15 serum-free medium (Lonza, Walkersville, MD, USA) in the presence of Dynabeads CD3-CD28 T cell expander (one bead per cell; Life Technologies, Carlsbad, CA, USA) and indicated cytokines: IL-1β (10 ng/mL), IL-6 (20 ng/mL), TGF-β (1 ng/mL) and IL-23 (100 ng/mL) (Miltenyi) for Th17 differentiation, as previously described [11 (link),21 (link)]. After 5–6 days, cells were harvested and stained for flow cytometry analysis, or extensively washed, counted, and re-stimulated 1 × 10⁶ cells/mL with Dynabeads CD3-CD28 T cell expander (one bead per cell) for 24 h for cytokine quantification.

PBMC were stained with the anti-CD4-FITC (Miltenyi; 1/100), and CD4⁺ T Lymphocytes were purified by immunomagnetic selection, using the anti-FITC isolation kit (Miltenyi, Bergisch Gladbach, Germany). After the isolation, the cells were stained with anti-CD4 FITC (Miltenyi; 1/100), anti-CD45RA BV421 (BD Biosciences, San Jose, CA, USA; 1/60), anti-CD45RO PE (BD Biosciences; 1/30), anti-CD27 APC (Miltenyi; 1/60), and CD4⁺ naive T-cells were sorted by a MoFlo high speed cell sorter (Beckman Coulter, Atlanta, GA, USA) as CD4^high, CD45RA^high, CD45RO⁻, and CD27⁺. Sorted cells had a purity of over 97%, measured by flow cytometry (data not shown). Naive CD4⁺ T-cells were cultured in 96-well round-bottomed plates (Corning) at a density of 5 × 10⁴ per well in X-VIVO 15 serum-free medium (Lonza, Walkersville, MD, USA) in the presence of a Dynabeads CD3-CD28 T-cell expander (one bead per cell; Life Technologies, Carlsbad, CA, USA). TGF-β (5 ng/mL), IL-4 (50 ng/mL) (Miltenyi), and OX40L (RnD, Minneapolis, MN, USA) were used to stimulate naïve CD4 T cells. After 6 days, supernatants were collected for IL-9 quantification.

T cells were purified from fresh whole blood from healthy, young volunteers by isopycnic centrifugation (Lymphoprep, Axis-Shield) and T cell negative selection (Dynal T cell negative selection kit, Invitrogen), as previously described [31 (link)]. Verbal consent to blood sampling was considered adequate by the local Ethics Committee, and was obtained. Biological specimens and all data obtained from their use for research were anonymized. Recruitment, verbal consent, and storage/use of blood specimens were carried out in accordance with the Declaration of Helsinki. T cells were added in the ratio 2.5 T cells: 1 RPE cell to the bottom or apical compartment as previously described with Dynabeads CD3/CD28 T Cell Expander (Invitrogen) [31 (link)]. This ratio was chosen to achieve cytokine concentrations in the supernatant comparable to concentrations in vivo in areas with high local T cell density. At the end of co-culture, media was gathered, and RPE cells were removed using a cell scraper. RNA was prepared as previously described [31 (link)]. A different donor was used for each independent replicate. Cell culture supernatants were diluted 1:5 or 1:10 in PBS-T with 5 mM EDTA for PTX3 protein quantification.

A total of 2.5 × 10⁵ PBMCs were seeded for 96 h in a U‐bottomed 96‐well plate with or without 10 and 25 μg/ml LEVs. Positive control was performed using Dynabeads CD3/CD28 T‐cell Expander (Invitrogen Life Technologies). The phenotype of CD4, CD8 and T cells was determined using Alexa Fluor405‐conjugated anti‐CD45RA, PE/Texas Red‐conjugated anti‐CD27, PerCP‐Cy5‐5‐conjugated anti‐CD4, APC‐Cy7‐conjugated anti‐CD8, PE‐conjugated anti‐TCR and FITC‐conjugated anti‐CD3 MAbs (all purchased by Miltenyi Biotec). After 20 min of staining at room temperature in the dark, cells were washed with FACS buffer (PBS supplemented with 2% FBS and 2 mM EDTA) and analysed by flow cytometry on a FACS Aria Flow Cytometer using FACS Diva software.