Anti cd49d clone 9f10

Cryopreserved PBMCs were thawed and stabilized overnight at 37°C. The cells were pre-incubated for 30 minutes at 37°C, 5% CO₂ with Polymyxin B (Sigma Aldrich) at a concentration of 10 μg/mL. The cells were then incubated for 16 hours with 100 μM of each recombinant protein or peptide in the presence of 2 μg/ml of anti-human CD28 (clone CD28.2, BD Biosciences) and anti-CD49d (clone 9F10, BioLegend) antibodies and IL-2 (50 IU/ml, Peprotech, Cranbury, NJ) and IL-7 (5 ng/ml, Peprotech). To detect intracellular staining, eBioscience™ Protein Transport Inhibitor Cocktail (500X, ThermoFisher Scientific) was added during the final 5 hours of culture. After 16 hours, the cells were labeled with Fixable Viability Stain 510 (BD Biosciences) for live cell staining and fluorophore conjugated anti-CD3 (clone SK7, BD Biosciences), CD4 (clone RPA-T4, BD Biosciences), CD8 (clone RPA-T8, BD Biosciences), Granzyme B (clone GB11, BD Biosciences), IFNγ (clone B27, BD Biosciences) and IL-17A (clone BL168, BD Biosciences) antibodies and detected using a BD LSR Fortessa.