Transcription factor fixation buffer

Blood was collected in sodium heparin tubes and processed within 3 h of collection. The whole blood assay was adapted from the protocol described by Hanekom et al.^{46 (link)} Briefly, 0.5 ml of whole blood was stimulated with a pool of 300 Mtb-derived peptides (Mtb300, 2 µg/ml)^{47 (link)} at 37 °C for 5 h in the presence of the costimulatory antibodies, anti-CD28 and anti-CD49d (1 μg/ml each; BD Biosciences) and Brefeldin-A (10 µg/ml; Sigma-Aldrich). Unstimulated cells were incubated with costimulatory antibodies and Brefeldin-A only. Red blood cells were then lysed in a 150 mM NH₄Cl, 10 mM KHCO₃, and 1 mM Na₄EDTA solution. Cells were stained with a Live/Dead Near-InfraRed dye (Invitrogen), and then fixed using a Transcription Factor Fixation buffer (eBioscience), cryopreserved in freezing media (50% fetal bovine serum, 40% RPMI, and 10% dimethyl sulfoxide) and stored in liquid nitrogen until use.

The assessment of the percentage of CD4 and CD8 T cells was performed on cryopreserved fixed whole blood as described⁵⁵ . Briefly bood was collected in sodium heparin tubes and processed within 3 h of collection. We adapted this assay to detect SARS-CoV-2 specific T cells using synthetic SARS-CoV-2 PepTivator peptides (Miltenyi Biotec, Surrey, UK), consisting of 15-mer sequences with 11 amino acid overlap covering the immunodominant parts of the spike (S) protein, and the complete sequence of the nucleocapsid (N) and membrane (M) proteins⁵⁵ . All peptides were combined in a single pool and used at a final concentration of 1 µg/ml. Briefly, 400 µl whole blood was stimulated with the SARS-CoV-2 S, N and M protein peptide pool at 37 °C for 5 h in the presence of co-stimulatory antibodies against CD28 (clone 28.2) and CD49d (clone L25) (1 µg/ml each; BD Biosciences, San Jose, CA, USA) and Brefeldin-A (10 µg/ml, Sigma-Aldrich, St Louis, MO, USA). Unstimulated blood was incubated with co-stimulatory antibodies, Brefeldin-A and an equimolar amount of DMSO. Red blood cell lysis and white cell fixation was performed in a single step using a Transcription Factor Fixation buffer (eBioscience, San Diego, CA, USA) for 20 min. Cells were then cryopreserved in freezing media (50% fetal bovine serum, 40% RPMI and 10% dimethyl sulfoxide) and stored in liquid nitrogen until batched analysis.