Modulating PD1 Pathway in PBMC

Freshly isolated PBMCs were cultured in 24-well plates at 2 × 10⁶ cells/well in RPMI 1640 containing 1% penicillin/streptomycin (Sigma), L-Glutamine and 10% heat-inactivated human serum, with or without γ-Mtb CFP-10 + ESAT-6 (10 μg/ml) at 37 °C in a humidified 5% CO₂ atmosphere. To neutralize PD1, 10 μg/ml anti-PD1 antibody (BioLegend) was added to the cells in presence of the antigen. Isotype control antibody μg/ml (BioLegend) was added to some cells this served as a control well for PD1 specific inhibition. After 96 h, cell-free culture supernatants were collected, aliquoted and stored at − 70 °C until cytokine concentrations were measured by ELISA as published in [8 (link)]. Cells were washed and stained for surface and intracellular markers.