Flow Cytometric Analysis of T and B Cell Subsets

Frozen PBMCs were thawed at 37 °C and washed in 10 mL of RPMI containing FCS (10%) (Sigma Aldrich). The pelleted cells were re-suspended in PBS (1 × 10⁶ cells/mL), were stained with combinations of antibodies (Supplementary Table S1) for 30 min at 4 °C and followed by 2 washes with PBS. For intracellular transcription factor staining for regulatory and follicular helper T cell staining, cells were surface-stained (anti-human CD3, anti-human CD4) and fixed with Foxp3 Fix Perm solution (eBiosciences, San Diego, CA, USA) for 30 min. This was followed by washing the cells, permeabilization with diluted permeabilization buffer (eBiosciences) and staining with antibodies for anti-human foxp3 and anti-human bcl6 for 30 min at 4 °C followed by 2 washes with PBS.
Samples were acquired using a Cyan ADP flow cytometer (Dako, Glostrup, Denmark). Data analysis was done using Summit V 4.3 software. Spectral overlap when using more than one colour was corrected via compensation. Appropriate isotype controls were used for setting gates. The gating strategy used to identify the T cell subset has been shown in Figure S1; the gating strategy for B cell subset distribution has been published [35 (link),36 (link)]. The detailed methods for stimulation of PBMCs to induce cytokine production by CD4 T cells and staining for toxin expressing immune cells can be found in the Supplementary Methods.