Appropriate isolation kits

Healthy donors were recruited from AZ volunteers and all samples were taken following appropriate blood collection guidelines. All blood donor volunteers signed Informed Consent form and donation was approved by AstraZeneca Institutional review board and local Ethic committee (033–10). PBMCs were isolated from the blood by density-gradient centrifugation in Leucosep tubes (Greiner Bio-One) filled with Ficoll-Paque plus (GE Healthcare) or using LymphoPrep density gradient medium and SepMate tubes (Stem Cell Technologies) for the Dendritic cell work. All cells were purified by either negative or positive selection using the appropriate isolation kits (see below) according to the manufacturer’s instructions (Miltenyi Biotech).

For T cells isolation, mice were sacrificed at the designated days of experiments to remove spleen and lymph nodes. The splenocytes were obtained by macerating spleens with a plunger and passing the tissue homogenates through a 70μm cell strainer. Cells were washed 2-3 times by centrifugation at 500g at 4°C for 5 min with incomplete RPMI-1640 to obtain a single-cell suspension. Pan-T cells were isolated using Pan-T cells isolation kit (Miltenyi Biotec, USA) using magnetic columns as per the manufacturer’s instructions. As and when required, the CD4⁺ and CD8⁺ T cells were isolated by positive selection using appropriate isolation kits (Miltenyi Biotec, USA). For isolation of lymph nodes (LN) lymphocytes, the brachial, axillary, and inguinal nodes were harvested and pooled together and the cells were isolated by mechanical maceration as described above.