Anti human cd28 mab

Human or mouse CD1d monomers (5 μg/mL, NIH tetramer core facility) were coated onto 96-well ELISA plates (Thermo Scientific, Waltham, MA) along with anti-human CD28 mAb (2 μg/mL, BioLegend) for 2 hours at 37°C. Lipids (10 μg/mL) were then added and incubated for 16 hrs at 37°C. Twenty five thousand Jurkat 76.3E1 transfectant cells were added to each well and cultured for 6 hours. The upregulation of CD69 surface expression was analyzed by flow cytometry.

In vitro priming of T cell responses and evaluation of peptide immunogenicity was carried out as described. Briefly, monocytes were purified from PBMC obtained from HLA-A*02:01 healthy donors by using CD14 microbeads (Miltenyi) and cultured for one day in complete RPMI medium containing rhGM-CSF (Miltenyi; 10 ng/ml) and rhIL-4 (Inmunotools; 2 ng/ml) for their differentiation into dendritic cells (DC). Next, rhTNF (Miltenyi; 10 ng/ml), IL-1β (Immunotools; 10 ng/ml) and PGE2 (Sigma-Aldrich; 1 µM) were added for DC maturation. One day later, cells were collected, loaded with peptide (10 µg/mL) for 1 hour and washed 3 times. DC were co-cultured (ratio 1:30) with CD8 T cells purified from the CD14^- fraction using CD8 microbeads (Miltenyi), in complete medium containing anti-human CD28 mAb (Biolegend; 0.5 µg/mL). Cells were fed on days 3, 7, 10, 14 and 17 with culture medium containing IL-2 (Proleukin, 20U/ml).
NeoAg-specific responses were evaluated at day 21 of co-culture by using a human IFN-γ ELISPOT set (BD-Biosciences) following manufacturer’s instructions. Expanded CD8 T cells (5 x 10⁴/well) were stimulated with peptides (10 µg/mL) and DC (10⁴/well) for 24 h. Next, wells were washed, incubated with conjugate antibody and developed. Spot forming cells were counted as described above.