Tnfα efluor 450 mab11

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.

Cryopreserved cells were thawed, washed and permeabilised 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, San Diego, CA, USA), CD4 BV785 (OKT4; BioLegend), CD8 BV510 (RPA‐T8; BioLegend), CD27 PE‐Cy5 (1A4CD27; Beckman Coulter, Brea), HLA‐DR BV605 (L243; BioLegend), Killer cell Lectin‐like Receptor G1 (KLRG1) PerCP‐eFluor 710 (13F12F2; eBioscience), IFN‐γ BV711 (4S.B3; BioLegend), TNF‐α eFluor 450 (Mab11; BioLegend eBioscience), IL‐2 PE/Dazzle (MQ1‐17H12, BioLegend eBioscience) MIP‐1β Alexa Fluor 488 (#24006; R&D systems, Minneapolis, MN, USA) and CD153 (R&D116614; R&D systems). Samples were acquired on a BD LSR‐II and analysed using FlowJo (v9.9.6, FlowJo LCC, Ashland, OR, USA). 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 cut‐off of 30 events was used.