Transcription factor perm wash buffer

Cryopreserved cells were thawed, washed and permeabilized with a Transcription Factor perm/wash buffer (eBioscience). Cells were then stained at room temperature for 45 min with the following antibodies: CD3 BV650 (OKT3; Biolegend), CD4 BV785 (OKT4; Biolegend), CD8 BV510 (RPA-T8; Biolegend), CD27 PE-Cy5 (1A4CD27; Beckman Coulter), HLA-DR BV605 (L243; Biolegend), Killer cell Lectin-like Receptor G1 (KLRG1) PerCP-eFluor 710 (13F12F2, eBioscience), Eomes eFluor 660 (WD1928, eBioscience), IFNγ BV711 (4S.B3; Biolegend), TNFα eFluor 450 (Mab11; Biolegend), IL-2 PE/Dazzle (MQ1-17H12, Biolegend), and CD153 (R&D116614, R&D). Samples were acquired on a BD LSR-II and analyzed using FlowJo (v9.9.6, TreeStar). A positive cytokine response was defined as at least twice the background of unstimulated cells. To define the phenotype of Mtb300-specific cells, a cut-off of 30 events was used. The gating strategy is presented in Supplementary Fig. 5.

Cell staining was performed on cryopreserved cells that were thawed, washed, and permeabilized with a transcription factor perm/wash buffer (eBioscience, Thermo Fisher Scientific). Cells were then stained at room temperature for 45 minutes with antibodies for CD3 BV650 (OKT3, BioLegend), CD4 BV785 (OKT4, BioLegend), CD8 BV510 (RPA-8, BioLegend), CD19-BV750 (HIB19, BioLegend), CD45RA Alexa Fluor 488 (HI100, BioLegend), CD27 PE-Cy5 (1A4CD27, Beckman Coulter), CD38 APC (HIT2, BD Biosciences), HLA-DR BV605 (L243, BioLegend), Ki67 PerCP-Cy5.5 (B56, BD Biosciences), PD-1 PE (J105, eBioscience, Thermo Fisher Scientific), GrB BV421 (BG11, BD Biosciences), IFN-γ BV711 (4S.B3, BioLegend), TNF-α PE-Cy7 (MAB11, BioLegend), and IL-2 PE/Dazzle 594 (MQ1-17H12, BioLegend). Samples were acquired on a BD Biosciences LSR II and analyzed using FlowJo v9.9.6. A positive response was defined as any cytokine response that was at least twice the background of unstimulated cells. To define the phenotype of SARS-CoV-2–specific CD4⁺ T cells, a cutoff of 20 events was used.

Cryopreserved cells were thawed, washed, and permeabilized with a transcription factor perm/wash buffer (eBioscience). Cells were then stained at room temperature for 45 minutes with the following antibodies: CD3 BV650 (OKT3; BioLegend, San Diego, California), CD4 BV785 (OKT4; BioLegend), CD8 BV510 (RPA-T8; BioLegend), HLA-DR BV605 (L243; BioLegend), IFN-γ BV711 (4S.B3; BioLegend), TNF-α PE-Cy7 (Mab11; BioLegend eBioscience), and IL-2 PE/Dazzle (MQ1-17H12; BioLegend). Samples were acquired on a BD LSR-II and analyzed using FlowJo (version 10.8.1, FlowJo, Ashland, Oregon). The gating strategy is presented in Supplementary Figure 1. A positive cytokine response was defined as at least twice the background of unstimulated cells. To define the phenotype of Mtb300-specific CD4 T cells, a cutoff of 30 events was used [27 ].