Cd3 bv650 okt3

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, CA, USA), CD4 BV785 (OKT4; Biolegend), CD8 BV510 (RPA-T8; Biolegend), CD27 PE-Cy5 (1A4CD27; Beckman Coulter, Brea, CA, USA), HLA-DR BV605 (L243; Biolegend), Ki67 PerCPcy5.5. (B56, BD), Granzyme B (GrB) BV421 (GB11, BD), Killer cell Lectin-like Receptor G1 (KLRG1) APC (13F12F2, eBioscience), IFN-γ BV711 (4S.B3; Biolegend), TNF-α PEcy7 (Mab11; Biolegend), IL-2 PE/Dazzle (MQ1-17H12, Biolegend), Mip-1β Alexa Fluor 488 (#24006, R&D systems, Minneapolis, MN, USA) and CD153 (R&D116614, R&D). Samples were acquired on a BD LSR-II and analyzed using FlowJo (v9.9.6, FlowJo LCC, Ashland, OR, USA). The granulocytes/monocytes population was defined based on their FSC/SSC characteristics. 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.

After co-culture with stimulator cells, PBMC were harvested, washed in 1× PBS, stained extracellularly, permeabilized, and stained intracellularly, using a 14-color panel containing Yellow Live/Dead viability kit (Invitrogen, Carlsbad, CA, USA) and mAb to CD14-BV570 (M5E2, Biolegend), CD19-BV570 (HIB19, Biolegend), CD3-BV650 (OKT3, Biolegend), CD4-PECy5 (RPA-T4, BD), CD8-PerCP-Cy5.5 (SK1, BD), CD45RA-biotin (HI100, BD), CD62L-APC-A780 (DREG-56, Ebioscience), integrin α₄β₇-A647 (ACT1; conjugated in house) streptavidin(SAV)-Qdot800 (Invitrogen) CD69-ECD (TP1.55.3, Beckman Coulter), IFN-γ-PE-Cy7 (B27, BD), TNF-α-A700 (MAb11, BD), IL-2-BV605 (MQ1-17H12, Biolegend), IL-17A-BV421 (BL168, Biolegend), and MIP-1β-PE (IC271P, R&D) as previously described (11 (link)). Samples were acquired using a customized LSRII flow cytometer (BD Biosciences) and analyzed using Winlist v7.0 (Verity Software House, Topsham, ME, USA). Absolute numbers of CD3, CD8, CD4 positive cells and CD8 memory subsets cells were calculated by using percentages obtained from flow cytometry analysis related to the absolute number of lymphocytes determined by routine blood count. Responses against S. Typhi were expressed as net percentage of positive cells (i.e., total percentage of positive cells in the presence of S. Typhi-infected targets minus percentage of positive cells in co-cultures with uninfected cells).

Cell staining was performed on cryopreserved fixed cells that were thawed and washed with 1% FACS washing buffer (1% FBS in PBS). A viability dye was not included during the staining process, as it has been reported that the omission of a viability dye during the staining process does not impact the detection and quantification of cytokines when using the whole blood-based T cell assay.⁸⁸ (link) Cells were stained with the following surface antibody markers: CD4 ECD (SFCI12T4D11, Beckman Coulter), CD8 BV510 (RPA-8, Biolegend) and incubated at room temperature for 20 min. Cells were permeabilized and stained with intracellular antibody markers CD3 BV650 (OKT3), IFN-γ AlexaFluor 700 (B27), TNF-α BV786 (Mab11) and IL-2 APC (MQ1-17H12) (all from Biolegend). Finally, cells were washed and fixed with Cellfix (BD Biosciences). Samples were acquired on a multiparameter BD Fortessa flow cytometer using Diva software version 9 and analyzed using FlowJo v10. Results are expressed as the frequency of CD4⁺ or CD8⁺ T cells expressing IFN-γ, TNF-α or IL-2. Cytokine responses presented are background subtracted values (from the frequency of cytokine produced by unstimulated cells).

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.